Polypeptides having cellulolytic enhancing activity and polynucleotides encoding same

ABSTRACT

The present invention relates to isolated polypeptides having cellulolytic enhancing activity and isolated polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a 35 U.S.C. 371 national application ofPCT/US2011/049692 filed on Aug. 30, 2011, which claims priority or thebenefit under 35 U.S.C. 119 of U.S. Provisional. Application No.61/378,320 filed on Aug. 30, 2011, the contents of which are fullyincorporated herein by reference.

STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSOREDRESEARCH AND DEVELOPMENT

This invention was made with Government support under CooperativeAgreement DE-FC36-08GO18080 awarded by the Department of Energy. Thegovernment has certain rights in this invention.

REFERENCE TO A SEQUENCE LISTING

This application contains a Sequence Listing in computer readable form,which is incorporated herein by reference.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to polypeptides having cellulolyticenhancing activity and polynucleotides encoding the polypeptides. Theinvention also relates to nucleic acid constructs, vectors, and hostcells comprising the polynucleotides as well as methods of producing andusing the polypeptides.

2. Description of the Related Art

Cellulose is a polymer of the simple sugar glucose covalently linked bybeta-1,4-bonds. Many microorganisms produce enzymes that hydrolyzebeta-linked glucans. These enzymes include endoglucanases,cellobiohydrolases, and beta-glucosidases. Endoglucanases digest thecellulose polymer at random locations, opening it to attack bycellobiohydrolases. Cellobiohydrolases sequentially release molecules ofcellobiose from the ends of the cellulose polymer. Cellobiose is awater-soluble beta-1,4-linked dimer of glucose. Beta-glucosidaseshydrolyze cellobiose to glucose.

The conversion of lignocellulosic feedstocks into ethanol has theadvantages of the ready availability of large amounts of feedstock, thedesirability of avoiding burning or land filling the materials, and thecleanliness of the ethanol fuel. Wood, agricultural residues, herbaceouscrops, and municipal solid wastes have been considered as feedstocks forethanol production. These materials primarily consist of cellulose,hemicellulose, and lignin. Once the lignocellulose is converted tofermentable sugars, e.g., glucose, the fermentable sugars are easilyfermented by yeast into ethanol.

There is a need in the art for new polypeptides having cellulolyticenhancing activity for use in the degradation of cellulosic materials.

The present invention provides polypeptides having cellulolyticenhancing activity and polynucleotides encoding the polypeptides.

SUMMARY OF THE INVENTION

The present invention relates to isolated polypeptides havingcellulolytic enhancing activity selected from the group consisting of:

(a) a polypeptide having at least 60% sequence identity to the maturepolypeptide of SEQ ID NO: 2, SEQ ID NO: 6, SEQ ID NO: 12, or SEQ ID NO:14; at least 65% sequence identity to the mature polypeptide of SEQ IDNO: 8 or SEQ ID NO: 10, or at least 75% sequence identity to the maturepolypeptide of SEQ ID NO: 4;

(b) a polypeptide encoded by a polynucleotide that hybridizes under atleast medium-high stringency conditions with (i) the mature polypeptidecoding sequence of SEQ ID NO: 1 or the cDNA sequence thereof, SEQ ID NO:3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, or SEQ IDNO: 13 or the cDNA sequence thereof, or (ii) the full-length complementof (i);

(c) a polypeptide encoded by a polynucleotide having at least 60%sequence identity to the mature polypeptide coding sequence of SEQ IDNO: 1 or the cDNA sequence thereof, SEQ ID NO: 5, SEQ ID NO: 11, or SEQID NO: 13 or the cDNA sequence thereof; at least 65% sequence identityto the mature polypeptide coding sequence of SEQ ID NO: 7 or SEQ ID NO:9, or at least 75% sequence identity to the mature polypeptide codingsequence of SEQ ID NO: 3;

(d) a variant of the mature polypeptide of SEQ ID NO: 2, SEQ ID NO: 4,SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, or SEQ ID NO:14 comprising a substitution, deletion, and/or insertion at one or more(e.g., several) positions; and

(e) a fragment of the polypeptide of (a), (b), (c), or (d) that hascellulolytic enhancing activity.

The present invention also relates to isolated polynucleotides encodingthe polypeptides of the present invention; nucleic acid constructs,recombinant expression vectors, and recombinant host cells comprisingthe polynucleotides; and methods of producing the polypeptides.

The present invention also relates to processes for degrading orconverting a cellulosic material, comprising: treating the cellulosicmaterial with an enzyme composition in the presence of a polypeptidehaving cellulolytic enhancing activity of the present invention.

The present invention also relates to processes of producing afermentation product, comprising: (a) saccharifying a cellulosicmaterial with an enzyme composition in the presence of a polypeptidehaving cellulolytic enhancing activity of the present invention; (b)fermenting the saccharified cellulosic material with one or more (e.g.,several) fermenting microorganisms to produce the fermentation product;and (c) recovering the fermentation product from the fermentation.

The present invention also relates to processes of fermenting acellulosic material, comprising: fermenting the cellulosic material withone or more (e.g., several) fermenting microorganisms, wherein thecellulosic material is saccharified with an enzyme composition in thepresence of a polypeptide having cellulolytic enhancing activity of thepresent invention.

The present invention also relates to a polynucleotide encoding a signalpeptide comprising or consisting of amino acids 1 to 20 of SEQ ID NO: 2,amino acids 1 to 25 of SEQ ID NO: 4, amino acids 1 to 20 of SEQ ID NO:6, amino acids 1 to 21 of SEQ ID NO: 8, amino acids 1 to 19 of SEQ IDNO: 10, amino acids 1 to 17 of SEQ ID NO: 12, or amino acids 1 to 20 ofSEQ ID NO: 14, which is operably linked to a gene encoding a protein,wherein the gene is foreign to the polynucleotide encoding the signalpeptide; nucleic acid constructs, expression vectors, and recombinanthost cells comprising the polynucleotides; and methods of producing aprotein.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows the effect of an Aspergillus aculeatus P23NHW GH61polypeptide (EXP03690) on enzymatic hydrolysis of pretreated corn stoverby a Trichoderma reesei cellulase preparation.

FIG. 2 shows the effect of an Aspergillus aculeatus P23NJS GH61polypeptide (EXP03691) on enzymatic hydrolysis of pretreated corn stoverby a Trichoderma reesei cellulase preparation.

FIG. 3 shows the effect of an Aspergillus aculeatus P23NNN GH61polypeptide (EXP03692) on enzymatic hydrolysis of pretreated corn stoverby a Trichoderma reesei cellulase preparation.

FIG. 4 shows the effect of an Aspergillus aculeatus P23NK1 GH61polypeptide (EXP03693) on enzymatic hydrolysis of pretreated corn stoverby a Trichoderma reesei cellulase preparation.

DEFINITIONS

Acetylxylan esterase: The term “acetylxylan esterase” means acarboxylesterase (EC 3.1.1.72) that catalyzes the hydrolysis of acetylgroups from polymeric xylan, acetylated xylose, acetylated glucose,alpha-napthyl acetate, and p-nitrophenyl acetate. For purposes of thepresent invention, acetylxylan esterase activity is determined using 0.5mM p-nitrophenylacetate as substrate in 50 mM sodium acetate pH 5.0containing 0.01% TWEEN™ 20 (polyoxyethylene sorbitan monolaurate). Oneunit of acetylxylan esterase is defined as the amount of enzyme capableof releasing 1 μmole of p-nitrophenolate anion per minute at pH 5, 25°C.

Allelic variant: The term “allelic variant” means any of two or morealternative forms of a gene occupying the same chromosomal locus.Allelic variation arises naturally through mutation, and may result inpolymorphism within populations. Gene mutations can be silent (no changein the encoded polypeptide) or may encode polypeptides having alteredamino acid sequences. An allelic variant of a polypeptide is apolypeptide encoded by an allelic variant of a gene.

Alpha-L-arabinofuranosidase: The term “alpha-L-arabinofuranosidase”means an alpha-L-arabinofuranoside arabinofuranohydrolase (EC 3.2.1.55)that catalyzes the hydrolysis of terminal non-reducingalpha-L-arabinofuranoside residues in alpha-L-arabinosides. The enzymeacts on alpha-L-arabinofuranosides, alpha-L-arabinans containing (1,3)-and/or (1,5)-linkages, arabinoxylans, and arabinogalactans.Alpha-L-arabinofuranosidase is also known as arabinosidase,alpha-arabinosidase, alpha-L-arabinosidase, alpha-arabinofuranosidase,polysaccharide alpha-L-arabinofuranosidase, alpha-L-arabinofuranosidehydrolase, L-arabinosidase, or alpha-L-arabinanase. For purposes of thepresent invention, alpha-L-arabinofuranosidase activity is determinedusing 5 mg of medium viscosity wheat arabinoxylan (MegazymeInternational Ireland, Ltd., Bray, Co. Wicklow, Ireland) per ml of 100mM sodium acetate pH 5 in a total volume of 200 μl for 30 minutes at 40°C. followed by arabinose analysis by AMINEX® HPX-87H columnchromatography (Bio-Rad Laboratories, Inc., Hercules, Calif., USA).

Alpha-glucuronidase: The term “alpha-glucuronidase” means analpha-D-glucosiduronate glucuronohydrolase (EC 3.2.1.139) that catalyzesthe hydrolysis of an alpha-D-glucuronoside to D-glucuronate and analcohol. For purposes of the present invention, alpha-glucuronidaseactivity is determined according to de Vries, 1998, J. Bacteriol. 180:243-249. One unit of alpha-glucuronidase equals the amount of enzymecapable of releasing 1 μmole of glucuronic or 4-O-methylglucuronic acidper minute at pH 5, 40° C.

Beta-glucosidase: The term “beta-glucosidase” means a beta-D-glucosideglucohydrolase (E.C. 3.2.1.21) that catalyzes the hydrolysis of terminalnon-reducing beta-D-glucose residues with the release of beta-D-glucose.For purposes of the present invention, beta-glucosidase activity isdetermined using p-nitrophenyl-beta-D-glucopyranoside as substrateaccording to the procedure of Venturi et al., 2002, Extracellularbeta-D-glucosidase from Chaetomium thermophilum var. coprophilum:production, purification and some biochemical properties, J. BasicMicrobiol. 42: 55-66. One unit of beta-glucosidase is defined as 1.0μmole of p-nitrophenolate anion produced per minute at 25° C., pH 4.8from 1 mM p-nitrophenyl-beta-D-glucopyranoside as substrate in 50 mMsodium citrate containing 0.01% TWEEN® 20.

Beta-xylosidase: The term “beta-xylosidase” means a beta-D-xylosidexylohydrolase (E.C. 3.2.1.37) that catalyzes the exo-hydrolysis of shortbeta→(4)-xylooligosaccharides to remove successive D-xylose residuesfrom non-reducing termini. For purposes of the present invention, oneunit of beta-xylosidase is defined as 1.0 μmole of p-nitrophenolateanion produced per minute at 40° C., pH 5 from 1 mMp-nitrophenyl-beta-D-xyloside as substrate in 100 mM sodium citratecontaining 0.01% TWEEN® 20.

cDNA: The term “cDNA” means a DNA molecule that can be prepared byreverse transcription from a mature, spliced, mRNA molecule obtainedfrom a eukaryotic or prokaryotic cell. cDNA lacks intron sequences thatmay be present in the corresponding genomic DNA. The initial, primaryRNA transcript is a precursor to mRNA that is processed through a seriesof steps, including splicing, before appearing as mature spliced mRNA.

Cellobiohydrolase: The term “cellobiohydrolase” means a1,4-beta-D-glucan cellobiohydrolase (E.C. 3.2.1.91) that catalyzes thehydrolysis of 1,4-beta-D-glucosidic linkages in cellulose,cellooligosaccharides, or any beta-1,4-linked glucose containingpolymer, releasing cellobiose from the reducing or non-reducing ends ofthe chain (Teeri, 1997, Crystalline cellulose degradation: New insightinto the function of cellobiohydrolases, Trends in Biotechnology 15:160-167; Teen et al., 1998, Trichoderma reesei cellobiohydrolases: whyso efficient on crystalline cellulose?, Biochem. Soc. Trans. 26:173-178). For purposes of the present invention, cellobiohydrolaseactivity is determined according to the procedures described by Lever etal., 1972, Anal. Biochem. 47: 273-279; van Tilbeurgh et al., 1982, FEBSLetters, 149: 152-156; van Tilbeurgh and Claeyssens, 1985, FEBS Letters,187: 283-288; and Tomme et al., 1988, Eur. J. Biochem. 170: 575-581. Inthe present invention, the Lever et al. method can be employed to assesshydrolysis of cellulose in corn stover, while the methods of vanTilbeurgh et al. and Tomme et al. can be used to determine thecellobiohydrolase activity on a fluorescent disaccharide derivative,4-methylumbelliferyl-beta-D-lactoside.

Cellulosic material: The term “cellulosic material” means any materialcontaining cellulose. The predominant polysaccharide in the primary cellwall of biomass is cellulose, the second most abundant is hemicellulose,and the third is pectin. The secondary cell wall, produced after thecell has stopped growing, also contains polysaccharides and isstrengthened by polymeric lignin covalently cross-linked tohemicellulose. Cellulose is a homopolymer of anhydrocellobiose and thusa linear beta-(1-4)-D-glucan, while hemicelluloses include a variety ofcompounds, such as xylans, xyloglucans, arabinoxylans, and mannans incomplex branched structures with a spectrum of substituents. Althoughgenerally polymorphous, cellulose is found in plant tissue primarily asan insoluble crystalline matrix of parallel glucan chains.Hemicelluloses usually hydrogen bond to cellulose, as well as to otherhemicelluloses, which help stabilize the cell wall matrix.

Cellulose is generally found, for example, in the stems, leaves, hulls,husks, and cobs of plants or leaves, branches, and wood of trees. Thecellulosic material can be, but is not limited to, agricultural residue,herbaceous material (including energy crops), municipal solid waste,pulp and paper mill residue, waste paper, and wood (including forestryresidue) (see, for example, Wiselogel et al., 1995, in Handbook onBioethanol (Charles E. Wyman, editor), pp. 105-118, Taylor & Francis,Washington D.C.; Wyman, 1994, Bioresource Technology 50: 3-16; Lynd,1990, Applied Biochemistry and Biotechnology 24/25: 695-719; Mosier etal., 1999, Recent Progress in Bioconversion of Lignocellulosics, inAdvances in Biochemical Engineering/Biotechnology, T. Scheper, managingeditor, Volume 65, pp. 23-40, Springer-Verlag, New York). It isunderstood herein that the cellulose may be in the form oflignocellulose, a plant cell wall material containing lignin, cellulose,and hemicellulose in a mixed matrix. In a preferred aspect, thecellulosic material is any biomass material. In another preferredaspect, the cellulosic material is lignocellulose, which comprisescellulose, hemicelluloses, and lignin.

In one aspect, the cellulosic material is agricultural residue. Inanother aspect, the cellulosic material is herbaceous material(including energy crops). In another aspect, the cellulosic material ismunicipal solid waste. In another aspect, the cellulosic material ispulp and paper mill residue. In another aspect, the cellulosic materialis waste paper. In another aspect, the cellulosic material is wood(including forestry residue).

In another aspect, the cellulosic material is arundo. In another aspect,the cellulosic material is bagasse. In another aspect, the cellulosicmaterial is bamboo. In another aspect, the cellulosic material is corncob. In another aspect, the cellulosic material is corn fiber. Inanother aspect, the cellulosic material is corn stover. In anotheraspect, the cellulosic material is miscanthus. In another aspect, thecellulosic material is orange peel. In another aspect, the cellulosicmaterial is rice straw. In another aspect, the cellulosic material isswitchgrass. In another aspect, the cellulosic material is wheat straw.

In another aspect, the cellulosic material is aspen. In another aspect,the cellulosic material is eucalyptus. In another aspect, the cellulosicmaterial is fir. In another aspect, the cellulosic material is pine. Inanother aspect, the cellulosic material is poplar. In another aspect,the cellulosic material is spruce. In another aspect, the cellulosicmaterial is willow.

In another aspect, the cellulosic material is algal cellulose. Inanother aspect, the cellulosic material is bacterial cellulose. Inanother aspect, the cellulosic material is cotton linter. In anotheraspect, the cellulosic material is filter paper. In another aspect, thecellulosic material is microcrystalline cellulose. In another aspect,the cellulosic material is phosphoric-acid treated cellulose.

In another aspect, the cellulosic material is an aquatic biomass. Asused herein the term “aquatic biomass” means biomass produced in anaquatic environment by a photosynthesis process. The aquatic biomass canbe algae, emergent plants, floating-leaf plants, or submerged plants.

The cellulosic material may be used as is or may be subjected topretreatment, using conventional methods known in the art, as describedherein. In a preferred aspect, the cellulosic material is pretreated.

Cellulolytic enzyme or cellulase: The term “cellulolytic enzyme” or“cellulase” means one or more (e.g., several) enzymes that hydrolyze acellulosic material. Such enzymes include endoglucanase(s),cellobiohydrolase(s), beta-glucosidase(s), or combinations thereof. Thetwo basic approaches for measuring cellulolytic activity include: (1)measuring the total cellulolytic activity, and (2) measuring theindividual cellulolytic activities (endoglucanases, cellobiohydrolases,and beta-glucosidases) as reviewed in Zhang et al., Outlook forcellulase improvement: Screening and selection strategies, 2006,Biotechnology Advances 24: 452-481. Total cellulolytic activity isusually measured using insoluble substrates, including Whatman NQ1filter paper, microcrystalline cellulose, bacterial cellulose, algalcellulose, cotton, pretreated lignocellulose, etc. The most common totalcellulolytic activity assay is the filter paper assay using Whatman No.1 filter paper as the substrate. The assay was established by theInternational Union of Pure and Applied Chemistry (IUPAC) (Ghose, 1987,Measurement of cellulase activities, Pure Appl. Chem. 59: 257-68).

For purposes of the present invention, cellulolytic enzyme activity isdetermined by measuring the increase in hydrolysis of a cellulosicmaterial by cellulolytic enzyme(s) under the following conditions: 1-50mg of cellulolytic enzyme protein/g of cellulose in PCS (or otherpretreated cellulosic material) for 3-7 days at a suitable temperature,e.g., 50° C., 55° C., or 60° C., compared to a control hydrolysiswithout addition of cellulolytic enzyme protein. Typical conditions are1 ml reactions, washed or unwashed PCS, 5% insoluble solids, 50 mMsodium acetate pH 5, 1 mM MnSO₄, 50° C., 55° C., or 60° C., 72 hours,sugar analysis by AMINEX® HPX-87H column (Bio-Rad Laboratories, Inc.,Hercules, Calif., USA).

Coding sequence: The term “coding sequence” means a polynucleotide,which directly specifies the amino acid sequence of a polypeptide. Theboundaries of the coding sequence are generally determined by an openreading frame, which begins with a start codon such as ATG, GTG, or TTGand ends with a stop codon such as TAA, TAG, or TGA. The coding sequencemay be a genomic DNA, cDNA, synthetic DNA, or a combination thereof.

Control sequences: The term “control sequences” means nucleic acidsequences necessary for expression of a polynucleotide encoding a maturepolypeptide of the present invention. Each control sequence may benative (i.e., from the same gene) or foreign (i.e., from a differentgene) to the polynucleotide encoding the polypeptide or native orforeign to each other. Such control sequences include, but are notlimited to, a leader, polyadenylation sequence, propeptide sequence,promoter, signal peptide sequence, and transcription terminator. At aminimum, the control sequences include a promoter, and transcriptionaland translational stop signals. The control sequences may be providedwith linkers for the purpose of introducing specific restriction sitesfacilitating ligation of the control sequences with the coding region ofthe polynucleotide encoding a polypeptide.

Endoglucanase: The term “endoglucanase” means anendo-1,4-(1,3;1,4)-beta-D-glucan 4-glucanohydrolase (E.C. 3.2.1.4) thatcatalyzes endohydrolysis of 1,4-beta-D-glycosidic linkages in cellulose,cellulose derivatives (such as carboxymethyl cellulose and hydroxyethylcellulose), lichenin, beta-1,4 bonds in mixed beta-1,3 glucans such ascereal beta-D-glucans or xyloglucans, and other plant materialcontaining cellulosic components. Endoglucanase activity can bedetermined by measuring reduction in substrate viscosity or increase inreducing ends determined by a reducing sugar assay (Zhang et al., 2006,Biotechnology Advances 24: 452-481). For purposes of the presentinvention, endoglucanase activity is determined using carboxymethylcellulose (CMC) as substrate according to the procedure of Ghose, 1987,Pure and Appl. Chem. 59: 257-268, at pH 5, 40° C.

Expression: The term “expression” includes any step involved in theproduction of a polypeptide including, but not limited to,transcription, post-transcriptional modification, translation,post-translational modification, and secretion.

Expression vector: The term “expression vector” means a linear orcircular DNA molecule that comprises a polynucleotide encoding apolypeptide and is operably linked to control sequences that provide forits expression.

Family 61 glycoside hydrolase: The term “Family 61 glycoside hydrolase”or “Family GH61” or “GH61” means a polypeptide falling into theglycoside hydrolase Family 61 according to Henrissat B., 1991, Aclassification of glycosyl hydrolases based on amino-acid sequencesimilarities, Biochem. J. 280: 309-316, and Henrissat B., and BairochA., 1996, Updating the sequence-based classification of glycosylhydrolases, Biochem. J. 316: 695-696. The enzymes in this family wereoriginally classified as a glycoside hydrolase family based onmeasurement of very weak endo-1,4-beta-D-glucanase activity in onefamily member. The structure and mode of action of these enzymes arenon-canonical and they cannot be considered as bona fide glycosidases.However, they are kept in the CAZy classification on the basis of theircapacity to enhance the breakdown of lignocellulose when used inconjunction with a cellulase or a mixture of cellulases.

Feruloyl esterase: The term “feruloyl esterase” means a4-hydroxy-3-methoxycinnamoyl-sugar hydrolase (EC 3.1.1.73) thatcatalyzes the hydrolysis of 4-hydroxy-3-methoxycinnamoyl (feruloyl)groups from esterified sugar, which is usually arabinose in “natural”substrates, to produce ferulate (4-hydroxy-3-methoxycinnamate). Feruloylesterase is also known as ferulic acid esterase, hydroxycinnamoylesterase, FAE-III, cinnamoyl ester hydrolase, FAEA, cinnAE, FAE-I, orFAE-II. For purposes of the present invention, feruloyl esteraseactivity is determined using 0.5 mM p-nitrophenylferulate as substratein 50 mM sodium acetate pH 5.0. One unit of feruloyl esterase equals theamount of enzyme capable of releasing 1 μmole of p-nitrophenolate anionper minute at pH 5, 25° C.

Fragment: The term “fragment” means a polypeptide having one or more(e.g., several) amino acids deleted from the amino and/or carboxylterminus of a mature polypeptide; wherein the fragment has cellulolyticenhancing activity. In one aspect, a fragment contains at least 280amino acid residues, e.g., at least 295 amino acid residues or at least310 amino acid residues of SEQ ID NO: 2. In another aspect, a fragmentcontains at least 310 amino acid residues, e.g., at least 330 amino acidresidues or at least 350 amino acid residues of SEQ ID NO: 4. In anotheraspect, a fragment contains at least 310 amino acid residues, e.g., atleast 330 amino acid residues or at least 350 amino acid residues of SEQID NO: 6. In another aspect, a fragment contains at least 320 amino acidresidues, e.g., at least 340 amino acid residues or at least 360 aminoacid residues of SEQ ID NO: 8. In another aspect, a fragment contains atleast 350 amino acid residues, e.g., at least 370 amino acid residues orat least 390 amino acid residues of SEQ ID NO: 10. In another aspect, afragment contains at least 220 amino acid residues, e.g., at least 230amino acid residues or at least 240 amino acid residues of SEQ ID NO:12. In another aspect, a fragment contains at least 220 amino acidresidues, e.g., at least 230 amino acid residues or at least 240 aminoacid residues of SEQ ID NO: 14.

Hemicellulolytic enzyme or hemicellulase: The term “hemicellulolyticenzyme” or “hemicellulase” means one or more (e.g., several) enzymesthat hydrolyze a hemicellulosic material. See, for example, Shallom, D.and Shoham, Y. Microbial hemicellulases. Current Opinion InMicrobiology, 2003, 6(3): 219-228). Hemicellulases are key components inthe degradation of plant biomass. Examples of hemicellulases include,but are not limited to, an acetylmannan esterase, an acetylxylanesterase, an arabinanase, an arabinofuranosidase, a coumaric acidesterase, a feruloyl esterase, a galactosidase, a glucuronidase, aglucuronoyl esterase, a mannanase, a mannosidase, a xylanase, and axylosidase. The substrates of these enzymes, the hemicelluloses, are aheterogeneous group of branched and linear polysaccharides that arebound via hydrogen bonds to the cellulose microfibrils in the plant cellwall, crosslinking them into a robust network. Hemicelluloses are alsocovalently attached to lignin, forming together with cellulose a highlycomplex structure. The variable structure and organization ofhemicelluloses require the concerted action of many enzymes for itscomplete degradation. The catalytic modules of hemicellulases are eitherglycoside hydrolases (GHs) that hydrolyze glycosidic bonds, orcarbohydrate esterases (CEs), which hydrolyze ester linkages of acetateor ferulic acid side groups. These catalytic modules, based on homologyof their primary sequence, can be assigned into GH and CE families. Somefamilies, with an overall similar fold, can be further grouped intoclans, marked alphabetically (e.g., GH-A). A most informative andupdated classification of these and other carbohydrate active enzymes isavailable in the Carbohydrate-Active Enzymes (CAZy) database.Hemicellulolytic enzyme activities can be measured according to Ghoseand Bisaria, 1987, Pure & Appl. Chem. 59: 1739-1752, at a suitabletemperature, e.g., 50° C., 55° C., or 60° C., and pH, e.g., 5.0 or 5.5.

High stringency conditions: The term “high stringency conditions” meansfor probes of at least 100 nucleotides in length, prehybridization andhybridization at 42° C. in 5×SSPE, 0.3% SDS, 200 micrograms/ml shearedand denatured salmon sperm DNA, and 50% formamide, following standardSouthern blotting procedures for 12 to 24 hours. The carrier material isfinally washed three times each for 15 minutes using 2×SSC, 0.2% SDS at65° C.

Host cell: The term “host cell” means any cell type that is susceptibleto transformation, transfection, transduction, or the like with anucleic acid construct or expression vector comprising a polynucleotideof the present invention. The term “host cell” encompasses any progenyof a parent cell that is not identical to the parent cell due tomutations that occur during replication.

Isolated: The term “isolated” means a substance in a form or environmentthat does not occur in nature. Non-limiting examples of isolatedsubstances include (1) any non-naturally occurring substance, (2) anysubstance including, but not limited to, any enzyme, variant, nucleicacid, protein, peptide or cofactor, that is at least partially removedfrom one or more or all of the naturally occurring constituents withwhich it is associated in nature; (3) any substance modified by the handof man relative to that substance found in nature; or (4) any substancemodified by increasing the amount of the substance relative to othercomponents with which it is naturally associated (e.g., multiple copiesof a gene encoding the substance; use of a stronger promoter than thepromoter naturally associated with the gene encoding the substance). Thepolypeptide of the present invention may be used in industrialapplications in the form of a fermentation broth product, that is, thepolypeptide of the present invention is a component of a fermentationbroth used as a product in industrial applications (e.g., ethanolproduction). The fermentation broth product will in addition to thepolypeptide of the present invention comprise additional ingredientsused in the fermentation process, such as, for example, cells(including, the host cells containing the gene encoding the polypeptideof the present invention which are used to produce the polypeptide ofinterest), cell debris, biomass, fermentation media and/or fermentationproducts. The fermentation broth may optionally be subjected to one ormore purification (including filtration) steps to remove or reduce onemore components of a fermentation process. Accordingly, an isolatedsubstance may be present in such a fermentation broth product.

Low stringency conditions: The term “low stringency conditions” meansfor probes of at least 100 nucleotides in length, prehybridization andhybridization at 42° C. in 5×SSPE, 0.3% SDS, 200 micrograms/ml shearedand denatured salmon sperm DNA, and 25% formamide, following standardSouthern blotting procedures for 12 to 24 hours. The carrier material isfinally washed three times each for 15 minutes using 2×SSC, 0.2% SDS at50° C.

Mature polypeptide: The term “mature polypeptide” means a polypeptide inits final form following translation and any post-translationalmodifications, such as N-terminal processing, C-terminal truncation,glycosylation, phosphorylation, etc. In one aspect, the maturepolypeptide is amino acids 21 to 344 of SEQ ID NO: 2 based on theSignalP program (Nielsen et al., 1997, Protein Engineering 10:1-6) thatpredicts amino acids 1 to 20 of SEQ ID NO: 2 are a signal peptide. Inanother aspect, the mature polypeptide is amino acids 26 to 400 of SEQID NO: 4 based on the SignalP program that predicts amino acids 1 to 25of SEQ ID NO: 4 are a signal peptide. In another aspect, the maturepolypeptide is amino acids 21 to 389 of SEQ ID NO: 6 based on theSignalP program that predicts amino acids 1 to 20 of SEQ ID NO: 6 are asignal peptide. In another aspect, the mature polypeptide is amino acids22 to 406 of SEQ ID NO: 8 based on the SignalP program that predictsamino acids 1 to 21 of SEQ ID NO: 8 are a signal peptide. In anotheraspect, the mature polypeptide is amino acids 20 to 427 of SEQ ID NO: 10based on the SignalP program that predicts amino acids 1 to 19 of SEQ IDNO: 10 are a signal peptide. In another aspect, the mature polypeptideis amino acids 18 to 267 of SEQ ID NO: 12 based on the SignalP programthat predicts amino acids 1 to 17 of SEQ ID NO: 12 are a signal peptide.In another aspect, the mature polypeptide is amino acids 21 to 273 ofSEQ ID NO: 14 based on the SignalP program that predicts amino acids 1to 20 of SEQ ID NO: 14 are a signal peptide. It is known in the art thata host cell may produce a mixture of two of more different maturepolypeptides (i.e., with a different C-terminal and/or N-terminal aminoacid) expressed by the same polynucleotide.

Mature polypeptide coding sequence: The term “mature polypeptide codingsequence” means a polynucleotide that encodes a mature polypeptidehaving cellulolytic enhancing activity. In one aspect, the maturepolypeptide coding sequence is nucleotides 61 to 1032 of SEQ ID NO: 1based on the SignalP program (Nielsen et al., 1997, supra) that predictsnucleotides 1 to 60 of SEQ ID NO: 1 encode a signal peptide. In anotheraspect, the mature polypeptide coding sequence is the cDNA sequence ofnucleotides 61 to 1032 of SEQ ID NO: 1. In another aspect, the maturepolypeptide coding sequence is nucleotides 76 to 1200 of SEQ ID NO: 3based on the SignalP program that predicts nucleotides 1 to 75 of SEQ IDNO: 3 encode a signal peptide. In another aspect, the mature polypeptidecoding sequence is nucleotides 61 to 1167 of SEQ ID NO: 5 based on theSignalP program that predicts nucleotides 1 to 60 of SEQ ID NO: 5 encodea signal peptide. In another aspect, the mature polypeptide codingsequence is nucleotides 64 to 1218 of SEQ ID NO: 7 based on the SignalPprogram that predicts nucleotides 1 to 63 of SEQ ID NO: 7 encode asignal peptide. In another aspect, the mature polypeptide codingsequence is nucleotides 58 to 1281 of SEQ ID NO: 9 based on the SignalPprogram that predicts nucleotides 1 to 57 of SEQ ID NO: 9 encode asignal peptide. In another aspect, the mature polypeptide codingsequence is nucleotides 52 to 801 of SEQ ID NO: 11 based on the SignalPprogram that predicts nucleotides 1 to 51 of SEQ ID NO: 11 encode asignal peptide. In another aspect, the mature polypeptide codingsequence is nucleotides 61 to 819 of SEQ ID NO: 13 based on the SignalPprogram that predicts nucleotides 1 to 60 of SEQ ID NO: 13 encode asignal peptide. In another aspect, the mature polypeptide codingsequence is the cDNA sequence of nucleotides 61 to 819 of SEQ ID NO: 13.

Medium stringency conditions: The term “medium stringency conditions”means for probes of at least 100 nucleotides in length, prehybridizationand hybridization at 42° C. in 5×SSPE, 0.3% SDS, 200 micrograms/mlsheared and denatured salmon sperm DNA, and 35% formamide, followingstandard Southern blotting procedures for 12 to 24 hours. The carriermaterial is finally washed three times each for 15 minutes using 2×SSC,0.2% SDS at 55° C.

Medium-high stringency conditions: The term “medium-high stringencyconditions” means for probes of at least 100 nucleotides in length,prehybridization and hybridization at 42° C. in 5×SSPE, 0.3% SDS, 200micrograms/ml sheared and denatured salmon sperm DNA, and 35% formamide,following standard Southern blotting procedures for 12 to 24 hours. Thecarrier material is finally washed three times each for 15 minutes using2×SSC, 0.2% SDS at 60° C.

Nucleic acid construct: The term “nucleic acid construct” means anucleic acid molecule, either single- or double-stranded, which isisolated from a naturally occurring gene or is modified to containsegments of nucleic acids in a manner that would not otherwise exist innature or which is synthetic, which comprises one or more controlsequences.

Operably linked: The term “operably linked” means a configuration inwhich a control sequence is placed at an appropriate position relativeto the coding sequence of a polynucleotide such that the controlsequence directs expression of the coding sequence.

Polypeptide having cellulolytic enhancing activity: The term“polypeptide having cellulolytic enhancing activity” means a GH61polypeptide that catalyzes the enhancement of the hydrolysis of acellulosic material by enzyme having cellulolytic activity. For purposesof the present invention, cellulolytic enhancing activity is determinedby measuring the increase in reducing sugars or the increase of thetotal of cellobiose and glucose from the hydrolysis of a cellulosicmaterial by cellulolytic enzyme under the following conditions: 1-50 mgof total protein/g of cellulose in PCS, wherein total protein iscomprised of 50-99.5% w/w cellulolytic enzyme protein and 0.5-50% w/wprotein of a GH61 polypeptide having cellulolytic enhancing activity for1-7 days at a suitable temperature, e.g., 50° C., 55° C., or 60° C., andpH, e.g., 5.0 or 5.5, compared to a control hydrolysis with equal totalprotein loading without cellulolytic enhancing activity (1-50 mg ofcellulolytic protein/g of cellulose in PCS). In a preferred aspect, amixture of CELLUCLAST® 1.5L (Novozymes NS, Bagsværd, Denmark) in thepresence of 2-3% of total protein weight Aspergillus oryzaebeta-glucosidase (recombinantly produced in Aspergillus oryzae accordingto WO 02/095014) or 2-3% of total protein weight Aspergillus fumigatusbeta-glucosidase (recombinantly produced in Aspergillus oryzae asdescribed in WO 2002/095014) of cellulase protein loading is used as thesource of the cellulolytic activity.

The GH61 polypeptides having cellulolytic enhancing activity enhance thehydrolysis of a cellulosic material catalyzed by enzyme havingcellulolytic activity by reducing the amount of cellulolytic enzymerequired to reach the same degree of hydrolysis preferably at least1.01-fold, e.g., at least 1.05-fold, at least 1.10-fold, at least1.25-fold, at least 1.5-fold, at least 2-fold, at least 3-fold, at least4-fold, at least 5-fold, at least 10-fold, or at least 20-fold.

The polypeptides of the present invention have at least 20%, e.g., atleast 40%, at least 50%, at least 60%, at least 70%, at least 80%, atleast 90%, at least 95%, and at least 100% of the cellulolytic enhancingactivity of the mature polypeptide of SEQ ID NO: 2, SEQ ID NO: 4, SEQ IDNO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, or SEQ ID NO: 14.

Pretreated corn stover: The term “PCS” or “Pretreated Corn Stover” meansa cellulosic material derived from corn stover by treatment with heatand dilute sulfuric acid.

Sequence identity: The relatedness between two amino acid sequences orbetween two nucleotide sequences is described by the parameter “sequenceidentity”.

For purposes of the present invention, the sequence identity between twoamino acid sequences is determined using the Needleman-Wunsch algorithm(Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453) as implementedin the Needle program of the EMBOSS package (EMBOSS: The EuropeanMolecular Biology Open Software Suite, Rice et al., 2000, Trends Genet.16: 276-277), preferably version 3.0.0, 5.0.0, or later. The parametersused are gap open penalty of 10, gap extension penalty of 0.5, and theEBLOSUM62 (EMBOSS version of BLOSUM62) substitution matrix. The outputof Needle labeled “longest identity” (obtained using the -nobriefoption) is used as the percent identity and is calculated as follows:(Identical Residues×100)/(Length of Alignment−Total Number of Gaps inAlignment)

For purposes of the present invention, the sequence identity between twodeoxyribonucleotide sequences is determined using the Needleman-Wunschalgorithm (Needleman and Wunsch, 1970, supra) as implemented in theNeedle program of the EMBOSS package (EMBOSS: The European MolecularBiology Open Software Suite, Rice et al., 2000, supra), preferablyversion 5.0.0 or later. The parameters used are gap open penalty of 10,gap extension penalty of 0.5, and the EDNAFULL (EMBOSS version of NCBINUC4.4) substitution matrix. The output of Needle labeled “longestidentity” (obtained using the -nobrief option) is used as the percentidentity and is calculated as follows:(Identical Deoxyribonucleotides×100)/(Length of Alignment−Total Numberof Gaps in Alignment)

Subsequence: The term “subsequence” means a polynucleotide having one ormore (e.g., several) nucleotides absent from the 5′ and/or 3′ end of amature polypeptide coding sequence; wherein the subsequence encodes afragment having cellulolytic enhancing activity. In one aspect, asubsequence contains at least 840 nucleotides, e.g., at least 885nucleotides or at least 930 nucleotides of SEQ ID NO: 1. In anotheraspect, a subsequence contains at least 930 nucleotides, e.g., at least990 nucleotides or at least 1050 nucleotides of SEQ ID NO: 3. In anotheraspect, a subsequence contains at least 930 nucleotides, e.g., at least990 nucleotides or at least 1050 nucleotides of SEQ ID NO: 5. In anotheraspect, a subsequence contains at least 960 nucleotides, e.g., at least1020 nucleotides or at least 1080 nucleotides of SEQ ID NO: 7. Inanother aspect, a subsequence contains at least 1050 nucleotides, e.g.,at least 1110 nucleotides or at least 1170 nucleotides of SEQ ID NO: 9.In another aspect, a subsequence contains at least 660 nucleotides,e.g., at least 690 nucleotides or at least 720 nucleotides of SEQ ID NO:11. In another aspect, a subsequence contains at least 660 nucleotides,e.g., at least 690 nucleotides or at least 720 nucleotides of SEQ ID NO:13.

Variant: The term “variant” means a polypeptide having cellulolyticenhancing activity comprising an alteration, i.e., a substitution,insertion, and/or deletion, at one or more (e.g., several) positions. Asubstitution means replacement of the amino acid occupying a positionwith a different amino acid; a deletion means removal of the amino acidoccupying a position; and an insertion means adding an amino acidadjacent to and immediately following the amino acid occupying aposition.

Xylan-containing material: The term “xylan-containing material” meansany material comprising a plant cell wall polysaccharide containing abackbone of beta-(1-4)-linked xylose residues. Xylans of terrestrialplants are heteropolymers possessing a beta-(1-4)-D-xylopyranosebackbone, which is branched by short carbohydrate chains. They compriseD-glucuronic acid or its 4-O-methyl ether, L-arabinose, and/or variousoligosaccharides, composed of D-xylose, L-arabinose, D- or L-galactose,and D-glucose. Xylan-type polysaccharides can be divided into homoxylansand heteroxylans, which include glucuronoxylans,(arabino)glucuronoxylans, (glucurono)arabinoxylans, arabinoxylans, andcomplex heteroxylans. See, for example, Ebringerova et al., 2005, Adv.Polym. Sci. 186: 1-67. In the methods of the present invention, anymaterial containing xylan may be used. In a preferred aspect, thexylan-containing material is lignocellulose.

Xylan degrading activity or xylanolytic activity: The term “xylandegrading activity” or “xylanolytic activity” means a biologicalactivity that hydrolyzes xylan-containing material. The two basicapproaches for measuring xylanolytic activity include: (1) measuring thetotal xylanolytic activity, and (2) measuring the individual xylanolyticactivities (e.g., endoxylanases, beta-xylosidases, arabinofuranosidases,alpha-glucuronidases, acetylxylan esterases, feruloyl esterases, andalpha-glucuronyl esterases). Recent progress in assays of xylanolyticenzymes was summarized in several publications including Biely andPuchard, Recent progress in the assays of xylanolytic enzymes, 2006,Journal of the Science of Food and Agriculture 86(11): 1636-1647;Spanikova and Biely, 2006, Glucuronoyl esterase—Novel carbohydrateesterase produced by Schizophyllum commune, FEBS Letters 580(19):4597-4601; Herrmann, Vrsanska, Jurickova, Hirsch, Biely, and Kubicek,1997, The beta-D-xylosidase of Trichoderma reesei is a multifunctionalbeta-D-xylan xylohydrolase, Biochemical Journal 321: 375-381.

Total xylan degrading activity can be measured by determining thereducing sugars formed from various types of xylan, including, forexample, oat spelt, beechwood, and larchwood xylans, or by photometricdetermination of dyed xylan fragments released from various covalentlydyed xylans. The most common total xylanolytic activity assay is basedon production of reducing sugars from polymeric 4-O-methylglucuronoxylan as described in Bailey, Biely, Poutanen, 1992,Interlaboratory testing of methods for assay of xylanase activity,Journal of Biotechnology 23(3): 257-270. Xylanase activity can also bedetermined with 0.2% AZCL-arabinoxylan as substrate in 0.01% TRITON®X-100 (4-(1,1,3,3-tetramethylbutyl)phenyl-polyethylene glycol) and 200mM sodium phosphate buffer pH 6 at 37° C. One unit of xylanase activityis defined as 1.0 μmole of azurine produced per minute at 37° C., pH 6from 0.2% AZCL-arabinoxylan as substrate in 200 mM sodium phosphate pH 6buffer.

For purposes of the present invention, xylan degrading activity isdetermined by measuring the increase in hydrolysis of birchwood xylan(Sigma Chemical Co., Inc., St. Louis, Mo., USA) by xylan-degradingenzyme(s) under the following typical conditions: 1 ml reactions, 5mg/ml substrate (total solids), 5 mg of xylanolytic protein/g ofsubstrate, 50 mM sodium acetate pH 5, 50° C., 24 hours, sugar analysisusing p-hydroxybenzoic acid hydrazide (PHBAH) assay as described byLever, 1972, A new reaction for colorimetric determination ofcarbohydrates, Anal. Biochem 47: 273-279.

Xylanase: The term “xylanase” means a 1,4-beta-D-xylan-xylohydrolase(E.C. 3.2.1.8) that catalyzes the endohydrolysis of 1,4-beta-D-xylosidiclinkages in xylans. For purposes of the present invention, xylanaseactivity is determined with 0.2% AZCL-arabinoxylan as substrate in 0.01%TRITON® X-100 and 200 mM sodium phosphate buffer pH 6 at 37° C. One unitof xylanase activity is defined as 1.0 μmole of azurine produced perminute at 37° C., pH 6 from 0.2% AZCL-arabinoxylan as substrate in 200mM sodium phosphate pH 6 buffer.

DETAILED DESCRIPTION OF THE INVENTION

Polypeptides Having Cellulolytic Enhancing Activity

In an embodiment, the present invention relates to isolated polypeptideshaving a sequence identity to the mature polypeptide of SEQ ID NO: 2,SEQ ID NO: 6, SEQ ID NO: 12, or SEQ ID NO: 14 of at least 60%, e.g., atleast 65%, at least 70%, at least 75%, at least 80%, at least 81%, atleast 82%, at least 83%, at least 84%, at least 85%, at least 86%, atleast 87%, at least 88%, at least 89%, at least 90%, at least 91%, atleast 92%, at least 93%, at least 94%, at least 95%, at least 96%, atleast 97%, at least 98%, at least 99%, or 100%; mature polypeptide ofSEQ ID NO: 8 or SEQ ID NO: 10 of at least 65%, e.g., at least 70%, atleast 75%, at least 80%, at least 81%, at least 82%, at least 83%, atleast 84%, at least 85%, at least 86%, at least 87%, at least 88%, atleast 89%, at least 90%, at least 91%, at least 92%, at least 93%, atleast 94%, at least 95%, at least 96%, at least 97%, at least 98%, atleast 99%, or 100%; or mature polypeptide of SEQ ID NO: 4 of at least75%, e.g., at least 80%, at least 81%, at least 82%, at least 83%, atleast 84%, at least 85%, at least 86%, at least 87%, at least 88%, atleast 89%, at least 90%, at least 91%, at least 92%, at least 93%, atleast 94%, at least 95%, at least 96%, at least 97%, at least 98%, atleast 99%, or 100%; which have cellulolytic enhancing activity. In oneaspect, the polypeptides differ by up to 10 amino acids, e.g., 1, 2, 3,4, 5, 6, 7, 8, 9, or 10, from the mature polypeptide of SEQ ID NO: 2,SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12,or SEQ ID NO: 14.

A polypeptide of the present invention preferably comprises or consistsof the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6,SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, or SEQ ID NO: 14; or anallelic variant thereof; or is a fragment thereof having cellulolyticenhancing activity. In another aspect, the polypeptide comprises orconsists of the mature polypeptide of SEQ ID NO: 2, SEQ ID NO: 4, SEQ IDNO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, or SEQ ID NO: 14. Inanother aspect, the polypeptide comprises or consists of amino acids 21to 344 of SEQ ID NO: 2. In another aspect, the polypeptide comprises orconsists of amino acids 26 to 400 of SEQ ID NO: 4. In another aspect,the polypeptide comprises or consists of amino acids 21 to 389 of SEQ IDNO: 6. In another aspect, the polypeptide comprises or consists of aminoacids 22 to 406 of SEQ ID NO: 8. In another aspect, the polypeptidecomprises or consists of amino acids 20 to 427 of SEQ ID NO: 10. Inanother aspect, the polypeptide comprises or consists of amino acids 18to 267 of SEQ ID NO: 12. In another aspect, the polypeptide comprises orconsists of amino acids 21 to 273 of SEQ ID NO: 14.

In another embodiment, the present invention relates to isolatedpolypeptides having cellulolytic enhancing activity that are encoded bypolynucleotides that hybridize under very low stringency conditions, lowstringency conditions, medium stringency conditions, medium-highstringency conditions, high stringency conditions, or very highstringency conditions with (i) the mature polypeptide coding sequence ofSEQ ID NO: 1 or the cDNA sequence thereof, SEQ ID NO: 3, SEQ ID NO: 5,SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, or SEQ ID NO: 13 or the cDNAsequence thereof, or (ii) the full-length complement of (i) (J.Sambrook, E. F. Fritsch, and T. Maniatis, 1989, Molecular Cloning, ALaboratory Manual, 2d edition, Cold Spring Harbor, N.Y.).

The polynucleotide of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ IDNO: 7, SEQ ID NO: 9, SEQ ID NO: 11, or SEQ ID NO: 13, or a subsequencethereof, as well as the amino acid sequence of SEQ ID NO: 2, SEQ ID NO:4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, or SEQ IDNO: 14, or a fragment thereof, may be used to design nucleic acid probesto identify and clone DNA encoding polypeptides having cellulolyticenhancing activity from strains of different genera or species accordingto methods well known in the art. In particular, such probes can be usedfor hybridization with the genomic DNA or cDNA of a cell of interest,following standard Southern blotting procedures, in order to identifyand isolate the corresponding gene therein. Such probes can beconsiderably shorter than the entire sequence, but should be at least15, e.g., at least 25, at least 35, or at least 70 nucleotides inlength. Preferably, the nucleic acid probe is at least 100 nucleotidesin length, e.g., at least 200 nucleotides, at least 300 nucleotides, atleast 400 nucleotides, at least 500 nucleotides, at least 600nucleotides, at least 700 nucleotides, at least 800 nucleotides, or atleast 900 nucleotides in length. Both DNA and RNA probes can be used.The probes are typically labeled for detecting the corresponding gene(for example, with ³²P, ³H, ³⁵S, biotin, or avidin). Such probes areencompassed by the present invention.

A genomic DNA or cDNA library prepared from such other strains may bescreened for DNA that hybridizes with the probes described above andencodes a polypeptide having cellulolytic enhancing activity. Genomic orother DNA from such other strains may be separated by agarose orpolyacrylamide gel electrophoresis, or other separation techniques. DNAfrom the libraries or the separated DNA may be transferred to andimmobilized on nitrocellulose or other suitable carrier material. Inorder to identify a clone or DNA that is homologous with SEQ ID NO: 1,SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11,or SEQ ID NO: 13, or a subsequence thereof, the carrier material is usedin a Southern blot.

For purposes of the present invention, hybridization indicates that thepolynucleotide hybridizes to a labeled nucleic acid probe correspondingto SEQ ID NO: 1 or the cDNA sequence thereof, SEQ ID NO: 3, SEQ ID NO:5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, or SEQ ID NO: 13; themature polypeptide coding sequence of SEQ ID NO: 1, SEQ ID NO: 3, SEQ IDNO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, or SEQ ID NO: 13 orthe cDNA sequence thereof; the full-length complement thereof; or asubsequence thereof; under very low to very high stringency conditions.Molecules to which the nucleic acid probe hybridizes under theseconditions can be detected using, for example, X-ray film or any otherdetection means known in the art.

In one aspect, the nucleic acid probe is the mature polypeptide codingsequence of SEQ ID NO: 1 or the cDNA sequence thereof, SEQ ID NO: 3, SEQID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, or SEQ ID NO: 13 orthe cDNA sequence thereof. In another aspect, the nucleic acid probe isnucleotides 61 to 1032 of SEQ ID NO: 1, nucleotides 76 to 1200 of SEQ IDNO: 3, nucleotides 61 to 1167 of SEQ ID NO: 5, nucleotides 64 to 1218 ofSEQ ID NO: 7, nucleotides 58 to 1281 of SEQ ID NO: 9, nucleotides 52 to801 of SEQ ID NO: 11, or nucleotides 61 to 819 of SEQ ID NO: 13. Inanother aspect, the nucleic acid probe is a polynucleotide that encodesthe polypeptide of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO:8, SEQ ID NO: 10, SEQ ID NO: 12, or SEQ ID NO: 14; or the maturepolypeptide thereof; or a fragment thereof. In another aspect, thenucleic acid probe is SEQ ID NO: 1 or the cDNA sequence thereof, SEQ IDNO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, or SEQID NO: 13 or the cDNA sequence thereof.

In another embodiment, the present invention relates to isolatedpolypeptides having cellulolytic enhancing activity encoded bypolynucleotides having a sequence identity to the mature polypeptidecoding sequence of SEQ ID NO: 1 or the cDNA sequence thereof, SEQ ID NO:5, SEQ ID NO: 11, or SEQ ID NO: 13 or the cDNA sequence thereof, of atleast 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%,81%, at least 82%, at least 83%, at least 84%, at least 85%, at least86%, at least 87%, at least 88%, at least 89%, at least 90%, at least91%, at least 92%, at least 93%, at least 94%, at least 95%, at least96%, at least 97%, at least 98%, at least 99%, or 100%; the maturepolypeptide coding sequence of SEQ ID NO: 7 or SEQ ID NO: 9 of at least65%, e.g., at least 70%, at least 75%, at least 80%, at least 81%, atleast 82%, at least 83%, at least 84%, at least 85%, at least 86%, atleast 87%, at least 88%, at least 89%, at least 90%, at least 91%, atleast 92%, at least 93%, at least 94%, at least 95%, at least 96%, atleast 97%, at least 98%, at least 99%, or 100%; or the maturepolypeptide coding sequence of SEQ ID NO: 3 of at least 75%, e.g., atleast 80%, at least 85%, at least 90%, at least 91%, at least 92%, atleast 93%, at least 94%, at least 95%, at least 96%, at least 97%, atleast 98%, at least 99%, or 100%.

In another embodiment, the present invention relates to variants of themature polypeptide of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ IDNO: 8, SEQ ID NO: 10, SEQ ID NO: 12, or SEQ ID NO: 14 comprising asubstitution, deletion, and/or insertion at one or more (e.g., several)positions. In an embodiment, the number of amino acid substitutions,deletions and/or insertions introduced into the mature polypeptide ofSEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10,SEQ ID NO: 12, or SEQ ID NO: 14 is not more than 10, e.g., 1, 2, 3, 4,5, 6, 7, 8, 9, or 10. The, amino acid changes may be of a minor nature,that is conservative amino acid substitutions or insertions that do notsignificantly affect the folding and/or activity of the protein; smalldeletions, typically of 1-30 amino acids; small amino- orcarboxyl-terminal extensions, such as an amino-terminal methionineresidue; a small linker peptide of up to 20-25 residues; or a smallextension that facilitates purification by changing net charge oranother function, such as a poly-histidine tract, an antigenic epitopeor a binding domain.

Examples of conservative substitutions are within the groups of basicamino acids (arginine, lysine and histidine), acidic amino acids(glutamic acid and aspartic acid), polar amino acids (glutamine andasparagine), hydrophobic amino acids (leucine, isoleucine and valine),aromatic amino acids (phenylalanine, tryptophan and tyrosine), and smallamino acids (glycine, alanine, serine, threonine and methionine). Aminoacid substitutions that do not generally alter specific activity areknown in the art and are described, for example, by H. Neurath and R. L.Hill, 1979, In, The Proteins, Academic Press, New York. Commonsubstitutions are Ala/Ser, Val/Ile, Asp/Glu, Thr/Ser, Ala/Gly, Ala/Thr,Ser/Asn, Ala/Val, Ser/Gly, Tyr/Phe, Ala/Pro, Lys/Arg, Asp/Asn, Leu/Ile,Leu/Val, Ala/Glu, and Asp/Gly.

Alternatively, the amino acid changes are of such a nature that thephysico-chemical properties of the polypeptides are altered. Forexample, amino acid changes may improve the thermal stability of thepolypeptide, alter the substrate specificity, change the pH optimum, andthe like.

Essential amino acids in a polypeptide can be identified according toprocedures known in the art, such as site-directed mutagenesis oralanine-scanning mutagenesis (Cunningham and Wells, 1989, Science 244:1081-1085). In the latter technique, single alanine mutations areintroduced at every residue in the molecule, and the resultant mutantmolecules are tested for cellulolytic enhancing activity to identifyamino acid residues that are critical to the activity of the molecule.See also, Hilton et al., 1996, J. Biol. Chem. 271: 4699-4708. The activesite of the enzyme or other biological interaction can also bedetermined by physical analysis of structure, as determined by suchtechniques as nuclear magnetic resonance, crystallography, electrondiffraction, or photoaffinity labeling, in conjunction with mutation ofputative contact site amino acids. See, for example, de Vos et al.,1992, Science 255: 306-312; Smith et al., 1992, J. Mol. Biol. 224:899-904; Wlodaver et al., 1992, FEBS Lett. 309: 59-64. The identity ofessential amino acids can also be inferred from an alignment with arelated polypeptide.

Single or multiple amino acid substitutions, deletions, and/orinsertions can be made and tested using known methods of mutagenesis,recombination, and/or shuffling, followed by a relevant screeningprocedure, such as those disclosed by Reidhaar-Olson and Sauer, 1988,Science 241: 53-57; Bowie and Sauer, 1989, Proc. Natl. Acad. Sci. USA86: 2152-2156; WO 95/17413; or WO 95/22625. Other methods that can beused include error-prone PCR, phage display (e.g., Lowman et al., 1991,Biochemistry 30: 10832-10837; U.S. Pat. No. 5,223,409; WO 92/06204), andregion-directed mutagenesis (Derbyshire et al., 1986, Gene 46: 145; Neret al., 1988, DNA 7: 127).

Mutagenesis/shuffling methods can be combined with high-throughput,automated screening methods to detect activity of cloned, mutagenizedpolypeptides expressed by host cells (Ness et al., 1999, NatureBiotechnology 17: 893-896). Mutagenized DNA molecules that encode activepolypeptides can be recovered from the host cells and rapidly sequencedusing standard methods in the art. These methods allow the rapiddetermination of the importance of individual amino acid residues in apolypeptide.

The polypeptide may be a hybrid polypeptide in which a region of onepolypeptide is fused at the N-terminus or the C-terminus of a region ofanother polypeptide.

The polypeptide may be a fusion polypeptide or cleavable fusionpolypeptide in which another polypeptide is fused at the N-terminus orthe C-terminus of the polypeptide of the present invention. A fusionpolypeptide is produced by fusing a polynucleotide encoding anotherpolypeptide to a polynucleotide of the present invention. Techniques forproducing fusion polypeptides are known in the art, and include ligatingthe coding sequences encoding the polypeptides so that they are in frameand that expression of the fusion polypeptide is under control of thesame promoter(s) and terminator. Fusion polypeptides may also beconstructed using intein technology in which fusion polypeptides arecreated post-translationally (Cooper et al., 1993, EMBO J. 12:2575-2583; Dawson et al., 1994, Science 266: 776-779).

A fusion polypeptide can further comprise a cleavage site between thetwo polypeptides. Upon secretion of the fusion protein, the site iscleaved releasing the two polypeptides. Examples of cleavage sitesinclude, but are not limited to, the sites disclosed in Martin et al.,2003, J. Ind. Microbiol. Biotechnol. 3: 568-576; Svetina et al., 2000,J. Biotechnol. 76: 245-251; Rasmussen-Wilson et al., 1997, Appl.Environ. Microbiol. 63: 3488-3493; Ward et al., 1995, Biotechnology 13:498-503; and Contreras et al., 1991, Biotechnology 9: 378-381; Eaton etal., 1986, Biochemistry 25: 505-512; Collins-Racie et al., 1995,Biotechnology 13: 982-987; Carter et al., 1989, Proteins: Structure,Function, and Genetics 6: 240-248; and Stevens, 2003, Drug DiscoveryWorld 4: 35-48.

Sources of Polypeptides Having Cellulolytic Enhancing Activity

A polypeptide having cellulolytic enhancing activity of the presentinvention may be obtained from microorganisms of any genus. For purposesof the present invention, the term “obtained from” as used herein inconnection with a given source shall mean that the polypeptide encodedby a polynucleotide is produced by the source or by a strain in whichthe polynucleotide from the source has been inserted. In one aspect, thepolypeptide obtained from a given source is secreted extracellularly.

The polypeptide may be a fungal polypeptide. For example, thepolypeptide may be a yeast polypeptide such as a Candida, Kluyveromyces,Pichia, Saccharomyces, Schizosaccharomyces, or Yarrowia polypeptide; ora filamentous fungal polypeptide such as an Acremonium, Agaricus,Alternaria, Aspergillus, Aureobasidium, Botryospaeria, Ceriporiopsis,Chaetomidium, Chrysosporium, Claviceps, Cochliobolus, Coprinopsis,Coptotermes, Corynascus, Cryphonectria, Cryptococcus, Diplodia, Exidia,Filibasidium, Fusarium, Gibberella, Holomastigotoides, Humicola, Irpex,Lentinula, Leptospaeria, Magnaporthe, Melanocarpus, Meripilus, Mucor,Myceliophthora, Neocallimastix, Neurospora, Paecilomyces, Penicillium,Phanerochaete, Piromyces, Poitrasia, Pseudoplectania,Pseudotrichonympha, Rhizomucor, Schizophyllum, Scytalidium, Talaromyces,Thermoascus, Thielavia, Tolypocladium, Trichoderma, Trichophaea,Verticillium, Volvariella, or Xylaria polypeptide.

In another aspect, the polypeptide is a Saccharomyces carlsbergensis,Saccharomyces cerevisiae, Saccharomyces diastaticus, Saccharomycesdouglasii, Saccharomyces kluyveri, Saccharomyces norbensis, orSaccharomyces oviformis polypeptide.

In another aspect, the polypeptide is an Acremonium cellulolyticus,Aspergillus aculeatus, Aspergillus awamori, Aspergillus foetidus,Aspergillus fumigatus, Aspergillus japonicus, Aspergillus nidulans,Aspergillus niger, Aspergillus oryzae, Chrysosporium inops,Chrysosporium keratinophilum, Chrysosporium lucknowense, Chrysosporiummerdarium, Chrysosporium pannicola, Chrysosporium queenslandicum,Chrysosporium tropicum, Chrysosporium zonatum, Fusarium bactridioides,Fusarium cerealis, Fusarium crookwellense, Fusarium culmorum, Fusariumgraminearum, Fusarium graminum, Fusarium heterosporum, Fusarium negundi,Fusarium oxysporum, Fusarium reticulatum, Fusarium roseum, Fusariumsambucinum, Fusarium sarcochroum, Fusarium sporotrichioides, Fusariumsulphureum, Fusarium torulosum, Fusarium trichothecioides, Fusariumvenenatum, Humicola grisea, Humicola insolens, Humicola lanuginosa,Irpex lacteus, Mucor miehei, Myceliophthora thermophila, Neurosporacrassa, Penicillium funiculosum, Penicillium purpurogenum, Phanerochaetechrysosporium, Thielavia achromatica, Thielavia albomyces, Thielaviaalbopilosa, Thielavia australeinsis, Thielavia fimeti, Thielaviamicrospora, Thielavia ovispora, Thielavia peruviana, Thielavia setosa,Thielavia spededonium, Thielavia subthermophila, Thielavia terrestris,Trichoderma harzianum, Trichoderma koningii, Trichodermalongibrachiatum, Trichoderma reesei, or Trichoderma viride polypeptide.

In another aspect, the polypeptide is an Aspergillus aculeatuspolypeptide, e.g., a polypeptide obtained from Aspergillus aculeatus CBS172.66.

It will be understood that for the aforementioned species the inventionencompasses both the perfect and imperfect states, and other taxonomicequivalents, e.g., anamorphs, regardless of the species name by whichthey are known. Those skilled in the art will readily recognize theidentity of appropriate equivalents.

Strains of these species are readily accessible to the public in anumber of culture collections, such as the American Type CultureCollection (ATCC), Deutsche Sammlung von Mikroorganismen andZellkulturen GmbH (DSMZ), Centraalbureau Voor Schimmelcultures (CBS),and Agricultural Research Service Patent Culture Collection, NorthernRegional Research Center (NRRL).

The polypeptide may be identified and obtained from other sourcesincluding microorganisms isolated from nature (e.g., soil, composts,water, etc.) or DNA samples obtained directly from natural materials(e.g., soil, composts, water, etc.) using the above-mentioned probes.Techniques for isolating microorganisms and DNA directly from naturalhabitats are well known in the art. A polynucleotide encoding thepolypeptide may then be obtained by similarly screening a genomic DNA orcDNA library of another microorganism or mixed DNA sample. Once apolynucleotide encoding a polypeptide has been detected with theprobe(s), the polynucleotide can be isolated or cloned by utilizingtechniques that are known to those of ordinary skill in the art (see,e.g., Sambrook et al., 1989, supra).

Polynucleotides

The present invention also relates to isolated polynucleotides encodinga polypeptide of the present invention.

The techniques used to isolate or clone a polynucleotide encoding apolypeptide are known in the art and include isolation from genomic DNAor cDNA, or a combination thereof. The cloning of the polynucleotidesfrom genomic DNA can be effected, e.g., by using the well knownpolymerase chain reaction (PCR) or antibody screening of expressionlibraries to detect cloned DNA fragments with shared structuralfeatures. See, e.g., Innis et al., 1990, PCR: A Guide to Methods andApplication, Academic Press, New York. Other nucleic acid amplificationprocedures such as ligase chain reaction (LCR), ligation activatedtranscription (LAT) and polynucleotide-based amplification (NASBA) maybe used. The polynucleotides may be cloned from a strain of Aspergillusaculeatus, or a related organism and thus, for example, may be anallelic or species variant of the polypeptide encoding region of thepolynucleotide.

In another embodiment, the present invention relates to isolatedpolynucleotides comprising or consisting of polynucleotides having adegree of sequence identity to the mature polypeptide coding sequence ofSEQ ID NO: 1 or the cDNA sequence thereof, SEQ ID NO: 5, SEQ ID NO: 11,or SEQ ID NO: 13 or the cDNA sequence thereof, of at least 60%, e.g., atleast 65%, at least 70%, at least 75%, at least 80%, at least 81%, atleast 82%, at least 83%, at least 84%, at least 85%, at least 86%, atleast 87%, at least 88%, at least 89%, at least 90%, at least 91%, atleast 92%, at least 93%, at least 94%, at least 95%, at least 96%, atleast 97%, at least 98%, at least 99%, or 100%; the mature polypeptidecoding sequence of SEQ ID NO: 7 or SEQ ID NO: 9 of at least 65%, e.g.,at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, atleast 83%, at least 84%, at least 85%, at least 86%, at least 87%, atleast 88%, at least 89%, at least 90%, at least 91%, at least 92%, atleast 93%, at least 94%, at least 95%, at least 96%, at least 97%, atleast 98%, at least 99%, or 100%; or the mature polypeptide codingsequence of SEQ ID NO: 3 of at least 75%, e.g., at least 80%, at least81%, at least 82%, at least 83%, at least 84%, at least 85%, at least86%, at least 87%, at least 88%, at least 89%, at least 90%, at least91%, at least 92%, at least 93%, at least 94%, at least 95%, at least96%, at least 97%, at least 98%, at least 99%, or 100%; which encodepolypeptides having cellulolytic enhancing activity.

Modification of a polynucleotide encoding a polypeptide of the presentinvention may be necessary for synthesizing polypeptides substantiallysimilar to the polypeptide. The term “substantially similar” to thepolypeptide refers to non-naturally occurring forms of the polypeptide.These polypeptides may differ in some engineered way from thepolypeptide isolated from its native source, e.g., variants that differin specific activity, thermostability, pH optimum, or the like. Thevariants may be constructed on the basis of the polynucleotide presentedas the mature polypeptide coding sequence of SEQ ID NO: 1 or the cDNAsequence thereof, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO:9, SEQ ID NO: 11, or SEQ ID NO: 13 or the cDNA sequence thereof, e.g., asubsequence thereof, and/or by introduction of nucleotide substitutionsthat do not result in a change in the amino acid sequence of thepolypeptide, but which correspond to the codon usage of the hostorganism intended for production of the enzyme, or by introduction ofnucleotide substitutions that may give rise to a different amino acidsequence. For a general description of nucleotide substitution, see,e.g., Ford et al., 1991, Protein Expression and Purification 2: 95-107.

In another embodiment, the present invention relates to isolatedpolynucleotides encoding polypeptides of the present invention, whichhybridize under low stringency conditions, medium stringency conditions,medium-high stringency conditions, high stringency conditions, or veryhigh stringency conditions with (i) the mature polypeptide codingsequence of SEQ ID NO: 1 or the cDNA sequence thereof, SEQ ID NO: 3, SEQID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, or SEQ ID NO: 13 orthe cDNA sequence thereof, or (ii) the full-length complement of (i); orallelic variants and subsequences thereof (Sambrook et al., 1989,supra), as defined herein.

In one aspect, the polynucleotide comprises or consists of SEQ ID NO: 1,SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11,or SEQ ID NO: 13; or the mature polypeptide coding sequence thereof; ora subsequence thereof that encodes a fragment having cellulolyticenhancing activity.

Nucleic Acid Constructs

The present invention also relates to nucleic acid constructs comprisinga polynucleotide of the present invention operably linked to one or morecontrol sequences that direct the expression of the coding sequence in asuitable host cell under conditions compatible with the controlsequences.

A polynucleotide may be manipulated in a variety of ways to provide forexpression of the polypeptide. Manipulation of the polynucleotide priorto its insertion into a vector may be desirable or necessary dependingon the expression vector. The techniques for modifying polynucleotidesutilizing recombinant DNA methods are well known in the art.

The control sequence may be a promoter, a polynucleotide that isrecognized by a host cell for expression of a polynucleotide encoding apolypeptide of the present invention. The promoter containstranscriptional control sequences that mediate the expression of thepolypeptide. The promoter may be any polynucleotide that showstranscriptional activity in the host cell including mutant, truncated,and hybrid promoters, and may be obtained from genes encodingextracellular or intracellular polypeptides either homologous orheterologous to the host cell.

Examples of suitable promoters for directing transcription of thenucleic acid constructs of the present invention in a bacterial hostcell are the promoters obtained from the Bacillus amyloliquefaciensalpha-amylase gene (amyQ), Bacillus licheniformis alpha-amylase gene(amyL), Bacillus licheniformis penicillinase gene (penP), Bacillusstearothermophilus maltogenic amylase gene (amyM), Bacillus subtilislevansucrase gene (sacB), Bacillus subtilis xylA and xylB genes,Bacillus thuringiensis cryIIIA gene (Agaisse and Lereclus, 1994,Molecular Microbiology 13: 97-107), E. coli lac operon, E. coli trcpromoter (Egon et al., 1988, Gene 69: 301-315), Streptomyces coelicoloragarase gene (dagA), and prokaryotic beta-lactamase gene (Villa-Kamaroffet al., 1978, Proc. Natl. Acad. Sci. USA 75: 3727-3731), as well as thetac promoter (DeBoer et al., 1983, Proc. Natl. Acad. Sci. USA 80:21-25). Further promoters are described in “Useful proteins fromrecombinant bacteria” in Gilbert et al., 1980, Scientific American 242:74-94; and in Sambrook et al., 1989, supra. Examples of tandem promotersare disclosed in WO 99/43835.

Examples of suitable promoters for directing transcription of thenucleic acid constructs of the present invention in a filamentous fungalhost cell are promoters obtained from the genes for Aspergillus nidulansacetamidase, Aspergillus niger neutral alpha-amylase, Aspergillus nigeracid stable alpha-amylase, Aspergillus niger or Aspergillus awamoriglucoamylase (glaA), Aspergillus oryzae TAKA amylase, Aspergillus oryzaealkaline protease, Aspergillus oryzae triose phosphate isomerase,Fusarium oxysporum trypsin-like protease (WO 96/00787), Fusariumvenenatum amyloglucosidase (WO 00/56900), Fusarium venenatum Dana (WO00/56900), Fusarium venenatum Quinn (WO 00/56900), Rhizomucor mieheilipase, Rhizomucor miehei aspartic proteinase, Trichoderma reeseibeta-glucosidase, Trichoderma reesei cellobiohydrolase I, Trichodermareesei cellobiohydrolase II, Trichoderma reesei endoglucanase I,Trichoderma reesei endoglucanase II, Trichoderma reesei endoglucanaseIII, Trichoderma reesei endoglucanase V, Trichoderma reesei xylanase I,Trichoderma reesei xylanase II, Trichoderma reesei xylanase III,Trichoderma reesei beta-xylosidase, and Trichoderma reesei translationelongation factor, as well as the NA2-tpi promoter (a modified promoterfrom an Aspergillus neutral alpha-amylase gene in which the untranslatedleader has been replaced by an untranslated leader from an Aspergillustriose phosphate isomerase gene; non-limiting examples include modifiedpromoters from an Aspergillus niger neutral alpha-amylase gene in whichthe untranslated leader has been replaced by an untranslated leader froman Aspergillus nidulans or Aspergillus oryzae triose phosphate isomerasegene); and mutant, truncated, and hybrid promoters thereof. Otherpromoters are described in U.S. Pat. No. 6,011,147.

In a yeast host, useful promoters are obtained from the genes forSaccharomyces cerevisiae enolase (ENO-1), Saccharomyces cerevisiaegalactokinase (GAL1), Saccharomyces cerevisiae alcoholdehydrogenase/glyceraldehyde-3-phosphate dehydrogenase (ADH1, ADH2/GAP),Saccharomyces cerevisiae triose phosphate isomerase (TPI), Saccharomycescerevisiae metallothionein (CUP1), and Saccharomyces cerevisiae3-phosphoglycerate kinase. Other useful promoters for yeast host cellsare described by Romanos et al., 1992, Yeast 8: 423-488.

The control sequence may also be a transcription terminator, which isrecognized by a host cell to terminate transcription. The terminator isoperably linked to the 3′-terminus of the polynucleotide encoding thepolypeptide. Any terminator that is functional in the host cell may beused in the present invention.

Preferred terminators for bacterial host cells are obtained from thegenes for Bacillus clausii alkaline protease (aprH), Bacilluslicheniformis alpha-amylase (amyL), and Escherichia coli ribosomal RNA(rrnB).

Preferred terminators for filamentous fungal host cells are obtainedfrom the genes for Aspergillus nidulans acetamidase, Aspergillusnidulans anthranilate synthase, Aspergillus niger glucoamylase,Aspergillus niger alpha-glucosidase, Aspergillus oryzae TAKA amylase,Fusarium oxysporum trypsin-like protease, Trichoderma reeseibeta-glucosidase, Trichoderma reesei cellobiohydrolase I, Trichodermareesei cellobiohydrolase II, Trichoderma reesei endoglucanase I,Trichoderma reesei endoglucanase II, Trichoderma reesei endoglucanaseIII, Trichoderma reesei endoglucanase V, Trichoderma reesei xylanase I,Trichoderma reesei xylanase II, Trichoderma reesei xylanase III,Trichoderma reesei beta-xylosidase, and Trichoderma reesei translationelongation factor.

Preferred terminators for yeast host cells are obtained from the genesfor Saccharomyces cerevisiae enolase, Saccharomyces cerevisiaecytochrome C (CYC1), and Saccharomyces cerevisiaeglyceraldehyde-3-phosphate dehydrogenase. Other useful terminators foryeast host cells are described by Romanos et al., 1992, supra.

The control sequence may also be an mRNA stabilizer region downstream ofa promoter and upstream of the coding sequence of a gene which increasesexpression of the gene.

Examples of suitable mRNA stabilizer regions are obtained from aBacillus thuringiensis cryIIIA gene (WO 94/25612) and a Bacillussubtilis SP82 gene (Hue et al., 1995, Journal of Bacteriology 177:3465-3471).

The control sequence may also be a leader, a nontranslated region of anmRNA that is important for translation by the host cell. The leader isoperably linked to the 5′-terminus of the polynucleotide encoding thepolypeptide. Any leader that is functional in the host cell may be used.

Preferred leaders for filamentous fungal host cells are obtained fromthe genes for Aspergillus oryzae TAKA amylase and Aspergillus nidulanstriose phosphate isomerase.

Suitable leaders for yeast host cells are obtained from the genes forSaccharomyces cerevisiae enolase (ENO-1), Saccharomyces cerevisiae3-phosphoglycerate kinase, Saccharomyces cerevisiae alpha-factor, andSaccharomyces cerevisiae alcoholdehydrogenase/glyceraldehyde-3-phosphate dehydrogenase (ADH2/GAP).

The control sequence may also be a polyadenylation sequence, a sequenceoperably linked to the 3′-terminus of the polynucleotide and, whentranscribed, is recognized by the host cell as a signal to addpolyadenosine residues to transcribed mRNA. Any polyadenylation sequencethat is functional in the host cell may be used.

Preferred polyadenylation sequences for filamentous fungal host cellsare obtained from the genes for Aspergillus nidulans anthranilatesynthase, Aspergillus niger glucoamylase, Aspergillus nigeralpha-glucosidase Aspergillus oryzae TAKA amylase, and Fusariumoxysporum trypsin-like protease.

Useful polyadenylation sequences for yeast host cells are described byGuo and Sherman, 1995, Mol. Cellular. Biol. 15: 5983-5990.

The control sequence may also be a signal peptide coding region thatencodes a signal peptide linked to the N-terminus of a polypeptide anddirects the polypeptide into the cell's secretory pathway. The 5′-end ofthe coding sequence of the polynucleotide may inherently contain asignal peptide coding sequence naturally linked in translation readingframe with the segment of the coding sequence that encodes thepolypeptide. Alternatively, the 5′-end of the coding sequence maycontain a signal peptide coding sequence that is foreign to the codingsequence. A foreign signal peptide coding sequence may be required wherethe coding sequence does not naturally contain a signal peptide codingsequence. Alternatively, a foreign signal peptide coding sequence maysimply replace the natural signal peptide coding sequence in order toenhance secretion of the polypeptide. However, any signal peptide codingsequence that directs the expressed polypeptide into the secretorypathway of a host cell may be used.

Effective signal peptide coding sequences for bacterial host cells arethe signal peptide coding sequences obtained from the genes for BacillusNCIB 11837 maltogenic amylase, Bacillus licheniformis subtilisin,Bacillus licheniformis beta-lactamase, Bacillus stearothermophilusalpha-amylase, Bacillus stearothermophilus neutral proteases (nprT,nprS, nprM), and Bacillus subtilis prsA. Further signal peptides aredescribed by Simonen and Palva, 1993, Microbiological Reviews 57:109-137.

Effective signal peptide coding sequences for filamentous fungal hostcells are the signal peptide coding sequences obtained from the genesfor Aspergillus niger neutral amylase, Aspergillus niger glucoamylase,Aspergillus oryzae TAKA amylase, Humicola insolens cellulase, Humicolainsolens endoglucanase V, Humicola lanuginosa lipase, and Rhizomucormiehei aspartic proteinase.

Useful signal peptides for yeast host cells are obtained from the genesfor Saccharomyces cerevisiae alpha-factor and Saccharomyces cerevisiaeinvertase. Other useful signal peptide coding sequences are described byRomanos et al., 1992, supra.

The control sequence may also be a propeptide coding sequence thatencodes a propeptide positioned at the N-terminus of a polypeptide. Theresultant polypeptide is known as a proenzyme or propolypeptide (or azymogen in some cases). A propolypeptide is generally inactive and canbe converted to an active polypeptide by catalytic or autocatalyticcleavage of the propeptide from the propolypeptide. The propeptidecoding sequence may be obtained from the genes for Bacillus subtilisalkaline protease (aprE), Bacillus subtilis neutral protease (nprT),Myceliophthora thermophila laccase (WO 95/33836), Rhizomucor mieheiaspartic proteinase, and Saccharomyces cerevisiae alpha-factor.

Where both signal peptide and propeptide sequences are present, thepropeptide sequence is positioned next to the N-terminus of apolypeptide and the signal peptide sequence is positioned next to theN-terminus of the propeptide sequence.

It may also be desirable to add regulatory sequences that regulateexpression of the polypeptide relative to the growth of the host cell.Examples of regulatory sequences are those that cause expression of thegene to be turned on or off in response to a chemical or physicalstimulus, including the presence of a regulatory compound. Regulatorysequences in prokaryotic systems include the lac, tac, and trp operatorsystems. In yeast, the ADH2 system or GAL1 system may be used. Infilamentous fungi, the Aspergillus niger glucoamylase promoter,Aspergillus oryzae TAKA alpha-amylase promoter, and Aspergillus oryzaeglucoamylase promoter, Trichoderma reesei cellobiohydrolase I promoter,and Trichoderma reesei cellobiohydrolase II promoter may be used. Otherexamples of regulatory sequences are those that allow for geneamplification. In eukaryotic systems, these regulatory sequences includethe dihydrofolate reductase gene that is amplified in the presence ofmethotrexate, and the metallothionein genes that are amplified withheavy metals. In these cases, the polynucleotide encoding thepolypeptide would be operably linked to the regulatory sequence.

Expression Vectors

The present invention also relates to recombinant expression vectorscomprising a polynucleotide of the present invention, a promoter, andtranscriptional and translational stop signals. The various nucleotideand control sequences may be joined together to produce a recombinantexpression vector that may include one or more convenient restrictionsites to allow for insertion or substitution of the polynucleotideencoding the polypeptide at such sites. Alternatively, thepolynucleotide may be expressed by inserting the polynucleotide or anucleic acid construct comprising the polynucleotide into an appropriatevector for expression. In creating the expression vector, the codingsequence is located in the vector so that the coding sequence isoperably linked with the appropriate control sequences for expression.

The recombinant expression vector may be any vector (e.g., a plasmid orvirus) that can be conveniently subjected to recombinant DNA proceduresand can bring about expression of the polynucleotide. The choice of thevector will typically depend on the compatibility of the vector with thehost cell into which the vector is to be introduced. The vector may be alinear or closed circular plasmid.

The vector may be an autonomously replicating vector, i.e., a vectorthat exists as an extrachromosomal entity, the replication of which isindependent of chromosomal replication, e.g., a plasmid, anextrachromosomal element, a minichromosome, or an artificial chromosome.The vector may contain any means for assuring self-replication.Alternatively, the vector may be one that, when introduced into the hostcell, is integrated into the genome and replicated together with thechromosome(s) into which it has been integrated. Furthermore, a singlevector or plasmid or two or more vectors or plasmids that togethercontain the total DNA to be introduced into the genome of the host cell,or a transposon, may be used.

The vector preferably contains one or more selectable markers thatpermit easy selection of transformed, transfected, transduced, or thelike cells. A selectable marker is a gene the product of which providesfor biocide or viral resistance, resistance to heavy metals, prototrophyto auxotrophs, and the like.

Examples of bacterial selectable markers are Bacillus licheniformis orBacillus subtilis dal genes, or markers that confer antibioticresistance such as ampicillin, chloramphenicol, kanamycin, neomycin,spectinomycin, or tetracycline resistance. Suitable markers for yeasthost cells include, but are not limited to, ADE2, HIS3, LEU2, LYS2,MET3, TRP1, and URA3. Selectable markers for use in a filamentous fungalhost cell include, but are not limited to, adeA(phosphoribosylaminoimidazole-succinocarboxamide synthase), adeB(phosphor-ribosylaminoimidazole synthase), amdS (acetamidase), argB(ornithine carbamoyltransferase), bar (phosphinothricinacetyltransferase), hph (hygromycin phosphotransferase), niaD (nitratereductase), pyrG (orotidine-5′-phosphate decarboxylase), sC (sulfateadenyltransferase), and trpC (anthranilate synthase), as well asequivalents thereof. Preferred for use in an Aspergillus cell areAspergillus nidulans or Aspergillus oryzae amdS and pyrG genes and aStreptomyces hygroscopicus bar gene. Preferred for use in a Trichodermacell are adeA, adeB, amdS, hph, and pyrG genes.

The selectable marker may be a dual selectable marker system asdescribed in WO 2010/039889. In one aspect, the dual selectable markeris a hph-tk dual selectable marker system.

The vector preferably contains an element(s) that permits integration ofthe vector into the host cell's genome or autonomous replication of thevector in the cell independent of the genome.

For integration into the host cell genome, the vector may rely on thepolynucleotide's sequence encoding the polypeptide or any other elementof the vector for integration into the genome by homologous ornon-homologous recombination. Alternatively, the vector may containadditional polynucleotides for directing integration by homologousrecombination into the genome of the host cell at a precise location(s)in the chromosome(s). To increase the likelihood of integration at aprecise location, the integrational elements should contain a sufficientnumber of nucleic acids, such as 100 to 10,000 base pairs, 400 to 10,000base pairs, and 800 to 10,000 base pairs, which have a high degree ofsequence identity to the corresponding target sequence to enhance theprobability of homologous recombination. The integrational elements maybe any sequence that is homologous with the target sequence in thegenome of the host cell. Furthermore, the integrational elements may benon-encoding or encoding polynucleotides. On the other hand, the vectormay be integrated into the genome of the host cell by non-homologousrecombination.

For autonomous replication, the vector may further comprise an origin ofreplication enabling the vector to replicate autonomously in the hostcell in question. The origin of replication may be any plasmidreplicator mediating autonomous replication that functions in a cell.The term “origin of replication” or “plasmid replicator” means apolynucleotide that enables a plasmid or vector to replicate in vivo.

Examples of bacterial origins of replication are the origins ofreplication of plasmids pBR322, pUC19, pACYC177, and pACYC184 permittingreplication in E. coli, and pUB110, pE194, pTA1060, and pAMβ1 permittingreplication in Bacillus.

Examples of origins of replication for use in a yeast host cell are the2 micron origin of replication, ARS1, ARS4, the combination of ARS1 andCEN3, and the combination of ARS4 and CEN6.

Examples of origins of replication useful in a filamentous fungal cellare AMA1 and ANS1 (Gems et al., 1991, Gene 98: 61-67; Cullen et al.,1987, Nucleic Acids Res. 15: 9163-9175; WO 00/24883). Isolation of theAMA1 gene and construction of plasmids or vectors comprising the genecan be accomplished according to the methods disclosed in WO 00/24883.

More than one copy of a polynucleotide of the present invention may beinserted into a host cell to increase production of a polypeptide. Anincrease in the copy number of the polynucleotide can be obtained byintegrating at least one additional copy of the sequence into the hostcell genome or by including an amplifiable selectable marker gene withthe polynucleotide where cells containing amplified copies of theselectable marker gene, and thereby additional copies of thepolynucleotide, can be selected for by cultivating the cells in thepresence of the appropriate selectable agent.

The procedures used to ligate the elements described above to constructthe recombinant expression vectors of the present invention are wellknown to one skilled in the art (see, e.g., Sambrook et al., 1989,supra).

Host Cells

The present invention also relates to recombinant host cells, comprisinga polynucleotide of the present invention operably linked to one or morecontrol sequences that direct the production of a polypeptide of thepresent invention. A construct or vector comprising a polynucleotide isintroduced into a host cell so that the construct or vector ismaintained as a chromosomal integrant or as a self-replicatingextra-chromosomal vector as described earlier. The term “host cell”encompasses any progeny of a parent cell that is not identical to theparent cell due to mutations that occur during replication. The choiceof a host cell will to a large extent depend upon the gene encoding thepolypeptide and its source.

The host cell may be any cell useful in the recombinant production of apolypeptide of the present invention, e.g., a prokaryote or a eukaryote.

The prokaryotic host cell may be any Gram-positive or Gram-negativebacterium. Gram-positive bacteria include, but are not limited to,Bacillus, Clostridium, Enterococcus, Geobacillus, Lactobacillus,Lactococcus, Oceanobacillus, Staphylococcus, Streptococcus, andStreptomyces. Gram-negative bacteria include, but are not limited to,Campylobacter, E. coli, Flavobacterium, Fusobacterium, Helicobacter,Ilyobacter, Neisseria, Pseudomonas, Salmonella, and Ureaplasma.

The bacterial host cell may be any Bacillus cell including, but notlimited to, Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillusbrevis, Bacillus circulans, Bacillus clausii, Bacillus coagulans,Bacillus firmus, Bacillus lautus, Bacillus lentus, Bacilluslicheniformis, Bacillus megaterium, Bacillus pumilus, Bacillusstearothermophilus, Bacillus subtilis, and Bacillus thuringiensis cells.

The bacterial host cell may also be any Streptococcus cell including,but not limited to, Streptococcus equisimilis, Streptococcus pyogenes,Streptococcus uberis, and Streptococcus equi subsp. Zooepidemicus cells.

The bacterial host cell may also be any Streptomyces cell including, butnot limited to, Streptomyces achromogenes, Streptomyces avermitilis,Streptomyces coelicolor, Streptomyces griseus, and Streptomyces lividanscells.

The introduction of DNA into a Bacillus cell may be effected byprotoplast transformation (see, e.g., Chang and Cohen, 1979, Mol. Gen.Genet. 168: 111-115), competent cell transformation (see, e.g., Youngand Spizizen, 1961, J. Bacteriol. 81: 823-829, or Dubnau andDavidoff-Abelson, 1971, J. Mol. Biol. 56: 209-221), electroporation(see, e.g., Shigekawa and Dower, 1988, Biotechniques 6: 742-751), orconjugation (see, e.g., Koehler and Thorne, 1987, J. Bacteriol. 169:5271-5278). The introduction of DNA into an E. coli cell may be effectedby protoplast transformation (see, e.g., Hanahan, 1983, J. Mol. Biol.166: 557-580) or electroporation (see, e.g., Dower et al., 1988, NucleicAcids Res. 16: 6127-6145). The introduction of DNA into a Streptomycescell may be effected by protoplast transformation, electroporation (see,e.g., Gong et al., 2004, Folia Microbiol. (Praha) 49: 399-405),conjugation (see, e.g., Mazodier et al., 1989, J. Bacteriol. 171:3583-3585), or transduction (see, e.g., Burke et al., 2001, Proc. Natl.Acad. Sci. USA 98: 6289-6294). The introduction of DNA into aPseudomonas cell may be effected by electroporation (see, e.g., Choi etal., 2006, J. Microbiol. Methods 64: 391-397) or conjugation (see, e.g.,Pinedo and Smets, 2005, Appl. Environ. Microbiol. 71: 51-57). Theintroduction of DNA into a Streptococcus cell may be effected by naturalcompetence (see, e.g., Perry and Kuramitsu, 1981, Infect. Immun. 32:1295-1297), protoplast transformation (see, e.g., Catt and Jollick,1991, Microbios 68: 189-207), electroporation (see, e.g., Buckley etal., 1999, Appl. Environ. Microbiol. 65: 3800-3804), or conjugation(see, e.g., Clewell, 1981, Microbiol. Rev. 45: 409-436). However, anymethod known in the art for introducing DNA into a host cell can beused.

The host cell may also be a eukaryote, such as a mammalian, insect,plant, or fungal cell.

The host cell may be a fungal cell. “Fungi” as used herein includes thephyla Ascomycota, Basidiomycota, Chytridiomycota, and Zygomycota as wellas the Oomycota and all mitosporic fungi (as defined by Hawksworth etal., In, Ainsworth and Bisby's Dictionary of The Fungi, 8th edition,1995, CAB International, University Press, Cambridge, UK).

The fungal host cell may be a yeast cell. “Yeast” as used hereinincludes ascosporogenous yeast (Endomycetales), basidiosporogenousyeast, and yeast belonging to the Fungi Imperfecti (Blastomycetes).Since the classification of yeast may change in the future, for thepurposes of this invention, yeast shall be defined as described inBiology and Activities of Yeast (Skinner, Passmore, and Davenport,editors, Soc. App. Bacteriol. Symposium Series No. 9, 1980).

The yeast host cell may be a Candida, Hansenula, Kluyveromyces, Pichia,Saccharomyces, Schizosaccharomyces, or Yarrowia cell, such as aKluyveromyces lactis, Saccharomyces carlsbergensis, Saccharomycescerevisiae, Saccharomyces diastaticus, Saccharomyces douglasii,Saccharomyces kluyveri, Saccharomyces norbensis, Saccharomycesoviformis, or Yarrowia lipolytica cell.

The fungal host cell may be a filamentous fungal cell. “Filamentousfungi” include all filamentous forms of the subdivision Eumycota andOomycota (as defined by Hawksworth et al., 1995, supra). The filamentousfungi are generally characterized by a mycelial wall composed of chitin,cellulose, glucan, chitosan, mannan, and other complex polysaccharides.Vegetative growth is by hyphal elongation and carbon catabolism isobligately aerobic. In contrast, vegetative growth by yeasts such asSaccharomyces cerevisiae is by budding of a unicellular thallus andcarbon catabolism may be fermentative.

The filamentous fungal host cell may be an Acremonium, Aspergillus,Aureobasidium, Bjerkandera, Ceriporiopsis, Chrysosporium, Coprinus,Coriolus, Cryptococcus, Filibasidium, Fusarium, Humicola, Magnaporthe,Mucor, Myceliophthora, Neocallimastix, Neurospora, Paecilomyces,Penicillium, Phanerochaete, Phlebia, Piromyces, Pleurotus,Schizophyllum, Talaromyces, Thermoascus, Thielavia, Tolypocladium,Trametes, or Trichoderma cell.

For example, the filamentous fungal host cell may be an Aspergillusawamori, Aspergillus foetidus, Aspergillus fumigatus, Aspergillusjaponicus, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae,Bjerkandera adusta, Ceriporiopsis aneirina, Ceriporiopsis caregiea,Ceriporiopsis gilvescens, Ceriporiopsis pannocinta, Ceriporiopsisrivulosa, Ceriporiopsis subrufa, Ceriporiopsis subvermispora,Chrysosporium inops, Chrysosporium keratinophilum, Chrysosporiumlucknowense, Chrysosporium merdarium, Chrysosporium pannicola,Chrysosporium queenslandicum, Chrysosporium tropicum, Chrysosporiumzonatum, Coprinus cinereus, Coriolus hirsutus, Fusarium bactridioides,Fusarium cerealis, Fusarium crookwellense, Fusarium culmorum, Fusariumgraminearum, Fusarium graminum, Fusarium heterosporum, Fusarium negundi,Fusarium oxysporum, Fusarium reticulatum, Fusarium roseum, Fusariumsambucinum, Fusarium sarcochroum, Fusarium sporotrichioides, Fusariumsulphureum, Fusarium torulosum, Fusarium trichothecioides, Fusariumvenenatum, Humicola insolens, Humicola lanuginosa, Mucor miehei,Myceliophthora thermophila, Neurospora crassa, Penicillium purpurogenum,Phanerochaete chrysosporium, Phlebia radiata, Pleurotus eryngii,Thielavia terrestris, Trametes villosa, Trametes versicolor, Trichodermaharzianum, Trichoderma koningii, Trichoderma longibrachiatum,Trichoderma reesei, or Trichoderma viride cell.

Fungal cells may be transformed by a process involving protoplastformation, transformation of the protoplasts, and regeneration of thecell wall in a manner known per se. Suitable procedures fortransformation of Aspergillus and Trichoderma host cells are describedin EP 238023, Yelton et al., 1984, Proc. Natl. Acad. Sci. USA 81:1470-1474, and Christensen et al., 1988, Bio/Technology 6: 1419-1422.Suitable methods for transforming Fusarium species are described byMalardier et al., 1989, Gene 78: 147-156, and WO 96/00787. Yeast may betransformed using the procedures described by Becker and Guarente, InAbelson, J. N. and Simon, M. I., editors, Guide to Yeast Genetics andMolecular Biology, Methods in Enzymology, Volume 194, pp 182-187,Academic Press, Inc., New York; Ito et al., 1983, J. Bacteriol. 153:163; and Hinnen et al., 1978, Proc. Natl. Acad. Sci. USA 75: 1920.

Methods of Production

The present invention also relates to methods of producing a polypeptideof the present invention, comprising: (a) cultivating a cell, which inits wild-type form produces the polypeptide, under conditions conducivefor production of the polypeptide; and (b) recovering the polypeptide.In one aspect, the cell is of the genus Aspergillus. In another aspect,the cell is Aspergillus aculeatus. In another aspect, the cell isAspergillus aculeatus CBS 172.66.

The present invention also relates to methods of producing a polypeptideof the present invention, comprising: (a) cultivating a recombinant hostcell of the present invention under conditions conducive for productionof the polypeptide; and (b) recovering the polypeptide.

The cells are cultivated in a nutrient medium suitable for production ofthe polypeptide using methods known in the art. For example, the cellsmay be cultivated by shake flask cultivation, or small-scale orlarge-scale fermentation (including continuous, batch, fed-batch, orsolid state fermentations) in laboratory or industrial fermentors in asuitable medium and under conditions allowing the polypeptide to beexpressed and/or isolated. The cultivation takes place in a suitablenutrient medium comprising carbon and nitrogen sources and inorganicsalts, using procedures known in the art. Suitable media are availablefrom commercial suppliers or may be prepared according to publishedcompositions (e.g., in catalogues of the American Type CultureCollection). If the polypeptide is secreted into the nutrient medium,the polypeptide can be recovered directly from the medium. If thepolypeptide is not secreted, it can be recovered from cell lysates.

The polypeptide may be detected using methods known in the art that arespecific for the polypeptides. These detection methods include, but arenot limited to, use of specific antibodies, formation of an enzymeproduct, or disappearance of an enzyme substrate. For example, an enzymeassay may be used to determine the activity of the polypeptide.

The polypeptide may be recovered using methods known in the art. Forexample, the polypeptide may be recovered from the nutrient medium byconventional procedures including, but not limited to, collection,centrifugation, filtration, extraction, spray-drying, evaporation, orprecipitation. In one aspect, the whole fermentation broth is recovered.

The polypeptide may be purified by a variety of procedures known in theart including, but not limited to, chromatography (e.g., ion exchange,affinity, hydrophobic, chromatofocusing, and size exclusion),electrophoretic procedures (e.g., preparative isoelectric focusing),differential solubility (e.g., ammonium sulfate precipitation),SDS-PAGE, or extraction (see, e.g., Protein Purification, Janson andRyden, editors, VCH Publishers, New York, 1989) to obtain substantiallypure polypeptides.

Plants

The present invention also relates to isolated plants, e.g., atransgenic plant, plant part, or plant cell, comprising a polynucleotideof the present invention so as to express and produce a polypeptide inrecoverable quantities. The polypeptide may be recovered from the plantor plant part. Alternatively, the plant or plant part containing thepolypeptide may be used as such for improving the quality of a food orfeed, e.g., improving nutritional value, palatability, and rheologicalproperties, or to destroy an antinutritive factor.

The transgenic plant can be dicotyledonous (a dicot) or monocotyledonous(a monocot). Examples of monocot plants are grasses, such as meadowgrass (blue grass, Poa), forage grass such as Festuca, Lolium, temperategrass, such as Agrostis, and cereals, e.g., wheat, oats, rye, barley,rice, sorghum, and maize (corn).

Examples of dicot plants are tobacco, legumes, such as lupins, potato,sugar beet, pea, bean and soybean, and cruciferous plants (familyBrassicaceae), such as cauliflower, rape seed, and the closely relatedmodel organism Arabidopsis thaliana.

Examples of plant parts are stem, callus, leaves, root, fruits, seeds,and tubers as well as the individual tissues comprising these parts,e.g., epidermis, mesophyll, parenchyme, vascular tissues, meristems.Specific plant cell compartments, such as chloroplasts, apoplasts,mitochondria, vacuoles, peroxisomes and cytoplasm are also considered tobe a plant part. Furthermore, any plant cell, whatever the tissueorigin, is considered to be a plant part. Likewise, plant parts such asspecific tissues and cells isolated to facilitate the utilization of theinvention are also considered plant parts, e.g., embryos, endosperms,aleurone and seed coats.

Also included within the scope of the present invention are the progenyof such plants, plant parts, and plant cells.

The transgenic plant or plant cell expressing the polypeptide may beconstructed in accordance with methods known in the art. In short, theplant or plant cell is constructed by incorporating one or moreexpression constructs encoding the polypeptide into the plant hostgenome or chloroplast genome and propagating the resulting modifiedplant or plant cell into a transgenic plant or plant cell.

The expression construct is conveniently a nucleic acid construct thatcomprises a polynucleotide encoding a polypeptide operably linked withappropriate regulatory sequences required for expression of thepolynucleotide in the plant or plant part of choice. Furthermore, theexpression construct may comprise a selectable marker useful foridentifying plant cells into which the expression construct has beenintegrated and DNA sequences necessary for introduction of the constructinto the plant in question (the latter depends on the DNA introductionmethod to be used).

The choice of regulatory sequences, such as promoter and terminatorsequences and optionally signal or transit sequences, is determined, forexample, on the basis of when, where, and how the polypeptide is desiredto be expressed. For instance, the expression of the gene encoding apolypeptide may be constitutive or inducible, or may be developmental,stage or tissue specific, and the gene product may be targeted to aspecific tissue or plant part such as seeds or leaves. Regulatorysequences are, for example, described by Tague et al., 1988, PlantPhysiology 86: 506.

For constitutive expression, the 35S-CaMV, the maize ubiquitin 1, or therice actin 1 promoter may be used (Franck et al., 1980, Cell 21:285-294; Christensen et al., 1992, Plant Mol. Biol. 18: 675-689; Zhanget al., 1991, Plant Cell 3: 1155-1165). Organ-specific promoters may be,for example, a promoter from storage sink tissues such as seeds, potatotubers, and fruits (Edwards and Coruzzi, 1990, Ann. Rev. Genet. 24:275-303), or from metabolic sink tissues such as meristems (Ito et al.,1994, Plant Mol. Biol. 24: 863-878), a seed specific promoter such asthe glutelin, prolamin, globulin, or albumin promoter from rice (Wu etal., 1998, Plant Cell Physiol. 39: 885-889), a Vicia faba promoter fromthe legumin B4 and the unknown seed protein gene from Vicia faba (Conradet al., 1998, J. Plant Physiol. 152: 708-711), a promoter from a seedoil body protein (Chen et al., 1998, Plant Cell Physiol. 39: 935-941),the storage protein napA promoter from Brassica napus, or any other seedspecific promoter known in the art, e.g., as described in WO 91/14772.Furthermore, the promoter may be a leaf specific promoter such as therbcs promoter from rice or tomato (Kyozuka et al., 1993, Plant Physiol.102: 991-1000), the chlorella virus adenine methyltransferase genepromoter (Mitra and Higgins, 1994, Plant Mol. Biol. 26: 85-93), the aldPgene promoter from rice (Kagaya et al., 1995, Mol. Gen. Genet. 248:668-674), or a wound inducible promoter such as the potato pin2 promoter(Xu et al., 1993, Plant Mol. Biol. 22: 573-588). Likewise, the promotermay be induced by abiotic treatments such as temperature, drought, oralterations in salinity or induced by exogenously applied substancesthat activate the promoter, e.g., ethanol, oestrogens, plant hormonessuch as ethylene, abscisic acid, and gibberellic acid, and heavy metals.

A promoter enhancer element may also be used to achieve higherexpression of a polypeptide in the plant. For instance, the promoterenhancer element may be an intron that is placed between the promoterand the polynucleotide encoding a polypeptide or domain. For instance,Xu et al., 1993, supra, disclose the use of the first intron of the riceactin 1 gene to enhance expression.

The selectable marker gene and any other parts of the expressionconstruct may be chosen from those available in the art.

The nucleic acid construct is incorporated into the plant genomeaccording to conventional techniques known in the art, includingAgrobacterium-mediated transformation, virus-mediated transformation,microinjection, particle bombardment, biolistic transformation, andelectroporation (Gasser et al., 1990, Science 244: 1293; Potrykus, 1990,Bio/Technology 8: 535; Shimamoto et al., 1989, Nature 338: 274).

Agrobacterium tumefaciens-mediated gene transfer is a method forgenerating transgenic dicots (for a review, see Hooykas andSchilperoort, 1992, Plant Mol. Biol. 19: 15-38) and for transformingmonocots, although other transformation methods may be used for theseplants. A method for generating transgenic monocots is particlebombardment (microscopic gold or tungsten particles coated with thetransforming DNA) of embryonic calli or developing embryos (Christou,1992, Plant J. 2: 275-281; Shimamoto, 1994, Curr. Opin. Biotechnol. 5:158-162; Vasil et al., 1992, Bio/Technology 10: 667-674). An alternativemethod for transformation of monocots is based on protoplasttransformation as described by Omirulleh et al., 1993, Plant Mol. Biol.21: 415-428. Additional transformation methods include those describedin U.S. Pat. Nos. 6,395,966 and 7,151,204 (both of which are hereinincorporated by reference in their entirety).

Following transformation, the transformants having incorporated theexpression construct are selected and regenerated into whole plantsaccording to methods well known in the art. Often the transformationprocedure is designed for the selective elimination of selection geneseither during regeneration or in the following generations by using, forexample, co-transformation with two separate T-DNA constructs or sitespecific excision of the selection gene by a specific recombinase.

In addition to direct transformation of a particular plant genotype witha construct of the present invention, transgenic plants may be made bycrossing a plant having the construct to a second plant lacking theconstruct. For example, a construct encoding a polypeptide can beintroduced into a particular plant variety by crossing, without the needfor ever directly transforming a plant of that given variety. Therefore,the present invention encompasses not only a plant directly regeneratedfrom cells which have been transformed in accordance with the presentinvention, but also the progeny of such plants. As used herein, progenymay refer to the offspring of any generation of a parent plant preparedin accordance with the present invention. Such progeny may include a DNAconstruct prepared in accordance with the present invention. Crossingresults in the introduction of a transgene into a plant line by crosspollinating a starting line with a donor plant line. Non-limitingexamples of such steps are described in U.S. Pat. No. 7,151,204.

Plants may be generated through a process of backcross conversion. Forexample, plants include plants referred to as a backcross convertedgenotype, line, inbred, or hybrid.

Genetic markers may be used to assist in the introgression of one ormore transgenes of the invention from one genetic background intoanother. Marker assisted selection offers advantages relative toconventional breeding in that it can be used to avoid errors caused byphenotypic variations. Further, genetic markers may provide dataregarding the relative degree of elite germplasm in the individualprogeny of a particular cross. For example, when a plant with a desiredtrait which otherwise has a non-agronomically desirable geneticbackground is crossed to an elite parent, genetic markers may be used toselect progeny which not only possess the trait of interest, but alsohave a relatively large proportion of the desired germplasm. In thisway, the number of generations required to introgress one or more traitsinto a particular genetic background is minimized.

The present invention also relates to methods of producing a polypeptideof the present invention comprising: (a) cultivating a transgenic plantor a plant cell comprising a polynucleotide encoding the polypeptideunder conditions conducive for production of the polypeptide; and (b)recovering the polypeptide.

Removal or Reduction of Cellulolytic Enhancing Activity

The present invention also relates to methods of producing a mutant of aparent cell, which comprises disrupting or deleting a polynucleotide, ora portion thereof, encoding a polypeptide of the present invention,which results in the mutant cell producing less of the polypeptide thanthe parent cell when cultivated under the same conditions.

The mutant cell may be constructed by reducing or eliminating expressionof the polynucleotide using methods well known in the art, for example,insertions, disruptions, replacements, or deletions. In a preferredaspect, the polynucleotide is inactivated. The polynucleotide to bemodified or inactivated may be, for example, the coding region or a partthereof essential for activity, or a regulatory element required forexpression of the coding region. An example of such a regulatory orcontrol sequence may be a promoter sequence or a functional partthereof, i.e., a part that is sufficient for affecting expression of thepolynucleotide. Other control sequences for possible modificationinclude, but are not limited to, a leader, polyadenylation sequence,propeptide sequence, signal peptide sequence, transcription terminator,and transcriptional activator.

Modification or inactivation of the polynucleotide may be performed bysubjecting the parent cell to mutagenesis and selecting for mutant cellsin which expression of the polynucleotide has been reduced oreliminated. The mutagenesis, which may be specific or random, may beperformed, for example, by use of a suitable physical or chemicalmutagenizing agent, by use of a suitable oligonucleotide, or bysubjecting the DNA sequence to PCR generated mutagenesis. Furthermore,the mutagenesis may be performed by use of any combination of thesemutagenizing agents.

Examples of a physical or chemical mutagenizing agent suitable for thepresent purpose include ultraviolet (UV) irradiation, hydroxylamine,N-methyl-N′-nitro-N-nitrosoguanidine (MNNG), O-methyl hydroxylamine,nitrous acid, ethyl methane sulphonate (EMS), sodium bisulphite, formicacid, and nucleotide analogues.

When such agents are used, the mutagenesis is typically performed byincubating the parent cell to be mutagenized in the presence of themutagenizing agent of choice under suitable conditions, and screeningand/or selecting for mutant cells exhibiting reduced or no expression ofthe gene.

Modification or inactivation of the polynucleotide may also beaccomplished by insertion, substitution, or deletion of one or morenucleotides in the gene or a regulatory element required fortranscription or translation thereof. For example, nucleotides may beinserted or removed so as to result in the introduction of a stop codon,the removal of the start codon, or a change in the open reading frame.Such modification or inactivation may be accomplished by site-directedmutagenesis or PCR generated mutagenesis in accordance with methodsknown in the art. Although, in principle, the modification may beperformed in vivo, i.e., directly on the cell expressing thepolynucleotide to be modified, it is preferred that the modification beperformed in vitro as exemplified below.

An example of a convenient way to eliminate or reduce expression of apolynucleotide is based on techniques of gene replacement, genedeletion, or gene disruption. For example, in the gene disruptionmethod, a nucleic acid sequence corresponding to the endogenouspolynucleotide is mutagenized in vitro to produce a defective nucleicacid sequence that is then transformed into the parent cell to produce adefective gene. By homologous recombination, the defective nucleic acidsequence replaces the endogenous polynucleotide. It may be desirablethat the defective polynucleotide also encodes a marker that may be usedfor selection of transformants in which the polynucleotide has beenmodified or destroyed. In an aspect, the polynucleotide is disruptedwith a selectable marker such as those described herein.

The present invention also relates to methods of inhibiting theexpression of a polypeptide having cellulolytic enhancing activity in acell, comprising administering to the cell or expressing in the cell adouble-stranded RNA (dsRNA) molecule, wherein the dsRNA comprises asubsequence of a polynucleotide of the present invention. In a preferredaspect, the dsRNA is about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 ormore duplex nucleotides in length.

The dsRNA is preferably a small interfering RNA (siRNA) or a micro RNA(miRNA). In a preferred aspect, the dsRNA is small interfering RNA forinhibiting transcription. In another preferred aspect, the dsRNA ismicro RNA for inhibiting translation.

The present invention also relates to such double-stranded RNA (dsRNA)molecules, comprising a portion of the mature polypeptide codingsequence of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQID NO: 9, SEQ ID NO: 11, or SEQ ID NO: 13 for inhibiting expression ofthe polypeptide in a cell. While the present invention is not limited byany particular mechanism of action, the dsRNA can enter a cell and causethe degradation of a single-stranded RNA (ssRNA) of similar or identicalsequences, including endogenous mRNAs. When a cell is exposed to dsRNA,mRNA from the homologous gene is selectively degraded by a processcalled RNA interference (RNAi).

The dsRNAs of the present invention can be used in gene-silencing. Inone aspect, the invention provides methods to selectively degrade RNAusing a dsRNAi of the present invention. The process may be practiced invitro, ex vivo or in vivo. In one aspect, the dsRNA molecules can beused to generate a loss-of-function mutation in a cell, an organ or ananimal. Methods for making and using dsRNA molecules to selectivelydegrade RNA are well known in the art; see, for example, U.S. Pat. Nos.6,489,127; 6,506,559; 6,511,824; and 6,515,109.

The present invention further relates to a mutant cell of a parent cellthat comprises a disruption or deletion of a polynucleotide encoding thepolypeptide or a control sequence thereof or a silenced gene encodingthe polypeptide, which results in the mutant cell producing less of thepolypeptide or no polypeptide compared to the parent cell.

The polypeptide-deficient mutant cells are particularly useful as hostcells for expression of native and heterologous polypeptides. Therefore,the present invention further relates to methods of producing a nativeor heterologous polypeptide, comprising: (a) cultivating the mutant cellunder conditions conducive for production of the polypeptide; and (b)recovering the polypeptide. The term “heterologous polypeptides” meanspolypeptides that are not native to the host cell, e.g., a variant of anative protein. The host cell may comprise more than one copy of apolynucleotide encoding the native or heterologous polypeptide.

The methods used for cultivation and purification of the product ofinterest may be performed by methods known in the art.

The methods of the present invention for producing an essentiallycellulolytic enhancing-free product is of particular interest in theproduction of eukaryotic polypeptides, in particular fungal proteinssuch as enzymes. The cellulolytic enhancing-deficient cells may also beused to express heterologous proteins of pharmaceutical interest such ashormones, growth factors, receptors, and the like. The term “eukaryoticpolypeptides” includes not only native polypeptides, but also thosepolypeptides, e.g., enzymes, which have been modified by amino acidsubstitutions, deletions or additions, or other such modifications toenhance activity, thermostability, pH tolerance and the like.

In a further aspect, the present invention relates to a protein productessentially free from cellulolytic enhancing activity that is producedby a method of the present invention.

Compositions

The present invention also relates to compositions comprising apolypeptide of the present invention. Preferably, the compositions areenriched in such a polypeptide. The term “enriched” indicates that thecellulolytic enhancing activity of the composition has been increased,e.g., with an enrichment factor of at least 1.1.

The compositions may comprise a polypeptide of the present invention asthe major enzymatic component, e.g., a mono-component composition.Alternatively, the compositions may comprise multiple enzymaticactivities, such as one or more (several) enzymes selected from thegroup consisting of a cellulase, a hemicellulase, an expansin, anesterase, a laccase, a ligninolytic enzyme, a pectinase, a peroxidase, aprotease, and a swollenin.

The compositions may be prepared in accordance with methods known in theart and may be in the form of a liquid or a dry composition. Thepolypeptide to be included in the composition may be stabilized inaccordance with methods known in the art.

The compositions may be a fermentation broth formulation or a cellcomposition, as described herein. Consequently, the present inventionalso relates to fermentation broth formulations and cell compositionscomprising a polypeptide having cellulolytic enhancing activity of thepresent invention. In some embodiments, the composition is a cell-killedwhole broth containing organic acid(s), killed cells and/or cell debris,and culture medium.

The term “fermentation broth” as used herein refers to a preparationproduced by cellular fermentation that undergoes no or minimal recoveryand/or purification. For example, fermentation broths are produced whenmicrobial cultures are grown to saturation, incubated undercarbon-limiting conditions to allow protein synthesis (e.g., expressionof enzymes by host cells) and secretion into cell culture medium. Thefermentation broth can contain unfractionated or fractionated contentsof the fermentation materials derived at the end of the fermentation.Typically, the fermentation broth is unfractionated and comprises thespent culture medium and cell debris present after the microbial cells(e.g., filamentous fungal cells) are removed, e.g., by centrifugation.In some embodiments, the fermentation broth contains spent cell culturemedium, extracellular enzymes, and viable and/or nonviable microbialcells.

In an embodiment, the fermentation broth formulation and cellcompositions comprise a first organic acid component comprising at leastone 1-5 carbon organic acid and/or a salt thereof and a second organicacid component comprising at least one 6 or more carbon organic acidand/or a salt thereof. In a specific embodiment, the first organic acidcomponent is acetic acid, formic acid, propionic acid, a salt thereof,or a mixture of two or more of the foregoing and the second organic acidcomponent is benzoic acid, cyclohexanecarboxylic acid, 4-methylvalericacid, phenylacetic acid, a salt thereof, or a mixture of two or more ofthe foregoing.

In one aspect, the composition contains an organic acid(s), andoptionally further contains killed cells and/or cell debris. In oneembodiment, the killed cells and/or cell debris are removed from acell-killed whole broth to provide a composition that is free of thesecomponents.

The fermentation broth formulations or cell compostions may furthercomprise a preservative and/or anti-microbial (e.g., bacteriostatic)agent, including, but not limited to, sorbitol, sodium chloride,potassium sorbate, and others known in the art.

The cell-killed whole broth or composition may further comprise one ormore enzyme activities such as cellobiohydrolase, endoglucanase,beta-glucosidase, endo-beta-1,3(4)-glucanase, glucohydrolase,xyloglucanase, xylanase, xylosidase, arabinofuranosidase,alpha-glucuronidase, acetyl xylan esterase, mannanase, mannosidase,alpha-galactosidase, mannan acetyl esterase, galactanase, arabinanase,pectate lyase, pectinase lyase, pectate lyase, polygalacturonase, pectinacetyl esterase, pectin methyl esterase, beta-galactosidase,galactanase, arabinanase, alpha-arabinofuranosidase,rhamnogalacturonase, ferrulic acid esterases rhamnogalacturonan lyase,rhamnogalacturonan acetyl esterase, xylogalacturonosidase,xylogalacturonase, rhamnogalacturonan lyase, lignin peroxidases,manganese-dependent peroxidases, hybrid peroxidases, with combinedproperties of lignin peroxidases and manganese-dependent peroxidases,glucoamylase, amylase, protease, and laccase.

In some embodiments, the cell-killed whole broth or composition includescellulolytic enzymes including, but not limited to, (i) endoglucanases(EG) or 1,4-D-glucan-4-glucanohydrolases (EC 3.2.1.4), (ii)exoglucanases, including 1,4-D-glucan glucanohydrolases (also known ascellodextnnases) (EC 3.2.1.74) and 1,4-D-glucan cellobiohydrolases(exo-cellobiohydrolases, CBH) (EC 3.2.1.91), and (iii) beta-glucosidase(BG) or beta-glucoside glucohydrolases (EC 3.2.1.21).

The cell-killed whole broth or composition may contain theunfractionated contents of the fermentation materials derived at the endof the fermentation. Typically, the cell-killed whole broth orcomposition contains the spent culture medium and cell debris presentafter the microbial cells (e.g., filamentous fungal cells) are grown tosaturation, incubated under carbon-limiting conditions to allow proteinsynthesis (e.g., expression of cellulase and/or glucosidase enzyme(s)).In some embodiments, the cell-killed whole broth or composition containsthe spent cell culture medium, extracellular enzymes, and killedfilamentous fungal cells. In some embodiments, the microbial cellspresent in the cell-killed whole broth or composition can bepermeabilized and/or lysed using methods known in the art.

A whole broth or cell composition as described herein is typically aliquid, but may contain insoluble components, such as killed cells, celldebris, culture media components, and/or insoluble enzyme(s). In someembodiments, insoluble components may be removed to provide a clarifiedliquid composition.

The whole broth formulations and cell compositions of the presentinvention may be produced by a method described in WO 90/15861 or WO2010/096673.

Examples are given below of preferred uses of the compositions of thepresent invention. The dosage of the composition and other conditionsunder which the composition is used may be determined on the basis ofmethods known in the art.

Uses

The present invention is also directed to the following processes forusing the polypeptides having cellulolytic enhancing activity, orcompositions thereof.

The present invention also relates to processes for degrading acellulosic material, comprising: treating the cellulosic material withan enzyme composition in the presence of a polypeptide havingcellulolytic enhancing activity of the present invention. In one aspect,the processes further comprise recovering the degraded or convertedcellulosic material. Soluble products of degradation or conversion ofthe cellulosic material can be separated from insoluble cellulosicmaterial using a method known in the art such as, for example,centrifugation, filtration, or gravity settling.

The present invention also relates to processes of producing afermentation product, comprising: (a) saccharifying a cellulosicmaterial with an enzyme composition in the presence of a polypeptidehaving cellulolytic enhancing activity of the present invention; (b)fermenting the saccharified cellulosic material with one or more (e.g.,several) fermenting microorganisms to produce the fermentation product;and (c) recovering the fermentation product from the fermentation.

The present invention also relates to processes of fermenting acellulosic material, comprising: fermenting the cellulosic material withone or more (e.g., several) fermenting microorganisms, wherein thecellulosic material is saccharified with an enzyme composition in thepresence of a polypeptide having cellulolytic enhancing activity of thepresent invention. In one aspect, the fermenting of the cellulosicmaterial produces a fermentation product. In another aspect, theprocesses further comprise recovering the fermentation product from thefermentation.

The processes of the present invention can be used to saccharify thecellulosic material to fermentable sugars and to convert the fermentablesugars to many useful fermentation products, e.g., fuel, potableethanol, and/or platform chemicals (e.g., acids, alcohols, ketones,gases, and the like). The production of a desired fermentation productfrom the cellulosic material typically involves pretreatment, enzymatichydrolysis (saccharification), and fermentation.

The processing of the cellulosic material according to the presentinvention can be accomplished using methods conventional in the art.Moreover, the processes of the present invention can be implementedusing any conventional biomass processing apparatus configured tooperate in accordance with the invention.

Hydrolysis (saccharification) and fermentation, separate orsimultaneous, include, but are not limited to, separate hydrolysis andfermentation (SHF); simultaneous saccharification and fermentation(SSF); simultaneous saccharification and co-fermentation (SSCF); hybridhydrolysis and fermentation (HHF); separate hydrolysis andco-fermentation (SHCF); hybrid hydrolysis and co-fermentation (HHCF);and direct microbial conversion (DMC), also sometimes calledconsolidated bioprocessing (CBP). SHF uses separate process steps tofirst enzymatically hydrolyze the cellulosic material to fermentablesugars, e.g., glucose, cellobiose, and pentose monomers, and thenferment the fermentable sugars to ethanol. In SSF, the enzymatichydrolysis of the cellulosic material and the fermentation of sugars toethanol are combined in one step (Philippidis, G. P., 1996, Cellulosebioconversion technology, in Handbook on Bioethanol: Production andUtilization, Wyman, C. E., ed., Taylor & Francis, Washington, D.C.,179-212). SSCF involves the co-fermentation of multiple sugars (Sheehan,J., and Himmel, M., 1999, Enzymes, energy and the environment: Astrategic perspective on the U.S. Department of Energy's research anddevelopment activities for bioethanol, Biotechnol. Prog. 15: 817-827).HHF involves a separate hydrolysis step, and in addition a simultaneoussaccharification and hydrolysis step, which can be carried out in thesame reactor. The steps in an HHF process can be carried out atdifferent temperatures, i.e., high temperature enzymaticsaccharification followed by SSF at a lower temperature that thefermentation strain can tolerate. DMC combines all three processes(enzyme production, hydrolysis, and fermentation) in one or more (e.g.,several) steps where the same organism is used to produce the enzymesfor conversion of the cellulosic material to fermentable sugars and toconvert the fermentable sugars into a final product (Lynd, L. R.,Weimer, P. J., van Zyl, W. H., and Pretorius, I. S., 2002, Microbialcellulose utilization: Fundamentals and biotechnology, Microbiol. Mol.Biol. Reviews 66: 506-577). It is understood herein that any methodknown in the art comprising pretreatment, enzymatic hydrolysis(saccharification), fermentation, or a combination thereof, can be usedin the practicing the processes of the present invention.

A conventional apparatus can include a fed-batch stirred reactor, abatch stirred reactor, a continuous flow stirred reactor withultrafiltration, and/or a continuous plug-flow column reactor (Fernandade Castilhos Corazza, Flávio Faria de Moraes, Gisella Maria Zanin andIvo Neitzel, 2003, Optimal control in fed-batch reactor for thecellobiose hydrolysis, Acta Scientiarum. Technology 25: 33-38; Gusakov,A. V., and Sinitsyn, A. P., 1985, Kinetics of the enzymatic hydrolysisof cellulose: 1. A mathematical model for a batch reactor process, Enz.Microb. Technol. 7: 346-352), an attrition reactor (Ryu, S. K., and Lee,J. M., 1983, Bioconversion of waste cellulose by using an attritionbioreactor, Biotechnol. Bioeng. 25: 53-65), or a reactor with intensivestirring induced by an electromagnetic field (Gusakov, A. V., Sinitsyn,A. P., Davydkin, I. Y., Davydkin, V. Y., Protas, O. V., 1996,Enhancement of enzymatic cellulose hydrolysis using a novel type ofbioreactor with intensive stirring induced by electromagnetic field,Appl. Biochem. Biotechnol. 56: 141-153). Additional reactor typesinclude fluidized bed, upflow blanket, immobilized, and extruder typereactors for hydrolysis and/or fermentation.

Pretreatment.

In practicing the processes of the present invention, any pretreatmentprocess known in the art can be used to disrupt plant cell wallcomponents of the cellulosic material (Chandra et al., 2007, Substratepretreatment: The key to effective enzymatic hydrolysis oflignocellulosics?, Adv. Biochem. Engin./Biotechnol. 108: 67-93; Galbeand Zacchi, 2007, Pretreatment of lignocellulosic materials forefficient bioethanol production, Adv. Biochem. Engin./Biotechnol. 108:41-65; Hendriks and Zeeman, 2009, Pretreatments to enhance thedigestibility of lignocellulosic biomass, Bioresource Technol. 100:10-18; Mosier et al., 2005, Features of promising technologies forpretreatment of lignocellulosic biomass, Bioresource Technol. 96:673-686; Taherzadeh and Karimi, 2008, Pretreatment of lignocellulosicwastes to improve ethanol and biogas production: A review, Int. J. ofMol. Sci. 9: 1621-1651; Yang and Wyman, 2008, Pretreatment: the key tounlocking low-cost cellulosic ethanol, Biofuels Bioproducts andBiorefining-Biofpr. 2: 26-40).

The cellulosic material can also be subjected to particle sizereduction, sieving, pre-soaking, wetting, washing, and/or conditioningprior to pretreatment using methods known in the art.

Conventional pretreatments include, but are not limited to, steampretreatment (with or without explosion), dilute acid pretreatment, hotwater pretreatment, alkaline pretreatment, lime pretreatment, wetoxidation, wet explosion, ammonia fiber explosion, organosolvpretreatment, and biological pretreatment. Additional pretreatmentsinclude ammonia percolation, ultrasound, electroporation, microwave,supercritical CO₂, supercritical H₂O, ozone, ionic liquid, and gammairradiation pretreatments.

The cellulosic material can be pretreated before hydrolysis and/orfermentation. Pretreatment is preferably performed prior to thehydrolysis. Alternatively, the pretreatment can be carried outsimultaneously with enzyme hydrolysis to release fermentable sugars,such as glucose, xylose, and/or cellobiose. In most cases thepretreatment step itself results in some conversion of biomass tofermentable sugars (even in absence of enzymes).

Steam Pretreatment. In steam pretreatment, the cellulosic material isheated to disrupt the plant cell wall components, including lignin,hemicellulose, and cellulose to make the cellulose and other fractions,e.g., hemicellulose, accessible to enzymes. The cellulosic material ispassed to or through a reaction vessel where steam is injected toincrease the temperature to the required temperature and pressure and isretained therein for the desired reaction time. Steam pretreatment ispreferably performed at 140-250° C., e.g., 160-200° C. or 170-190° C.,where the optimal temperature range depends on addition of a chemicalcatalyst. Residence time for the steam pretreatment is preferably 1-60minutes, e.g., 1-30 minutes, 1-20 minutes, 3-12 minutes, or 4-10minutes, where the optimal residence time depends on temperature rangeand addition of a chemical catalyst. Steam pretreatment allows forrelatively high solids loadings, so that the cellulosic material isgenerally only moist during the pretreatment. The steam pretreatment isoften combined with an explosive discharge of the material after thepretreatment, which is known as steam explosion, that is, rapid flashingto atmospheric pressure and turbulent flow of the material to increasethe accessible surface area by fragmentation (Duff and Murray, 1996,Bioresource Technology 855: 1-33; Galbe and Zacchi, 2002, Appl.Microbiol. Biotechnol. 59: 618-628; U.S. Patent Application No.20020164730). During steam pretreatment, hemicellulose acetyl groups arecleaved and the resulting acid autocatalyzes partial hydrolysis of thehemicellulose to monosaccharides and oligosaccharides. Lignin is removedto only a limited extent.

Chemical Pretreatment: The term “chemical treatment” refers to anychemical pretreatment that promotes the separation and/or release ofcellulose, hemicellulose, and/or lignin. Such a pretreatment can convertcrystalline cellulose to amorphous cellulose. Examples of suitablechemical pretreatment processes include, for example, dilute acidpretreatment, lime pretreatment, wet oxidation, ammonia fiber/freezeexplosion (AFEX), ammonia percolation (APR), ionic liquid, andorganosolv pretreatments.

A catalyst such as H₂SO₄ or SO₂ (typically 0.3 to 5% w/w) is often addedprior to steam pretreatment, which decreases the time and temperature,increases the recovery, and improves enzymatic hydrolysis (Ballesteroset al., 2006, Appl. Biochem. Biotechnol. 129-132: 496-508; Varga et al.,2004, Appl. Biochem. Biotechnol. 113-116: 509-523; Sassner et al., 2006,Enzyme Microb. Technol. 39: 756-762). In dilute acid pretreatment, thecellulosic material is mixed with dilute acid, typically H₂SO₄, andwater to form a slurry, heated by steam to the desired temperature, andafter a residence time flashed to atmospheric pressure. The dilute acidpretreatment can be performed with a number of reactor designs, e.g.,plug-flow reactors, counter-current reactors, or continuouscounter-current shrinking bed reactors (Duff and Murray, 1996, supra;Schell et al., 2004, Bioresource Technol. 91: 179-188; Lee et al., 1999,Adv. Biochem. Eng. Biotechnol. 65: 93-115).

Several methods of pretreatment under alkaline conditions can also beused. These alkaline pretreatments include, but are not limited to,sodium hydroxide, lime, wet oxidation, ammonia percolation (APR), andammonia fiber/freeze explosion (AFEX).

Lime pretreatment is performed with calcium oxide or calcium hydroxideat temperatures of 85-150° C. and residence times from 1 hour to severaldays (Wyman et al., 2005, Bioresource Technol. 96: 1959-1966; Mosier etal., 2005, Bioresource Technol. 96: 673-686). WO 2006/110891, WO2006/110899, WO 2006/110900, and WO 2006/110901 disclose pretreatmentmethods using ammonia.

Wet oxidation is a thermal pretreatment performed typically at 180-200°C. for 5-15 minutes with addition of an oxidative agent such as hydrogenperoxide or over-pressure of oxygen (Schmidt and Thomsen, 1998,Bioresource Technol. 64: 139-151; Palonen et al., 2004, Appl. Biochem.Biotechnol. 117: 1-17; Varga et al., 2004, Biotechnol. Bioeng. 88:567-574; Martin et al., 2006, J. Chem. Technol. Biotechnol. 81:1669-1677). The pretreatment is performed preferably at 1-40% drymatter, e.g., 2-30% dry matter or 5-20% dry matter, and often theinitial pH is increased by the addition of alkali such as sodiumcarbonate.

A modification of the wet oxidation pretreatment method, known as wetexplosion (combination of wet oxidation and steam explosion) can handledry matter up to 30%. In wet explosion, the oxidizing agent isintroduced during pretreatment after a certain residence time. Thepretreatment is then ended by flashing to atmospheric pressure (WO2006/032282).

Ammonia fiber explosion (AFEX) involves treating the cellulosic materialwith liquid or gaseous ammonia at moderate temperatures such as 90-150°C. and high pressure such as 17-20 bar for 5-10 minutes, where the drymatter content can be as high as 60% (Gollapalli et al., 2002, Appl.Biochem. Biotechnol. 98: 23-35; Chundawat et al., 2007, Biotechnol.Bioeng. 96: 219-231; Alizadeh et al., 2005, Appl. Biochem. Biotechnol.121: 1133-1141; Teymouri et al., 2005, Bioresource Technol. 96:2014-2018). During AFEX pretreatment cellulose and hemicelluloses remainrelatively intact. Lignin-carbohydrate complexes are cleaved.

Organosolv pretreatment delignifies the cellulosic material byextraction using aqueous ethanol (40-60% ethanol) at 160-200° C. for30-60 minutes (Pan et al., 2005, Biotechnol. Bioeng. 90: 473-481; Pan etal., 2006, Biotechnol. Bioeng. 94: 851-861; Kurabi et al., 2005, Appl.Biochem. Biotechnol. 121: 219-230). Sulphuric acid is usually added as acatalyst. In organosolv pretreatment, the majority of hemicellulose andlignin is removed.

Other examples of suitable pretreatment methods are described by Schellet al., 2003, Appl. Biochem. and Biotechnol. Vol. 105-108, p. 69-85, andMosier et al., 2005, Bioresource Technology 96: 673-686, and U.S.Published Application 2002/0164730.

In one aspect, the chemical pretreatment is preferably carried out as adilute acid treatment, and more preferably as a continuous dilute acidtreatment. The acid is typically sulfuric acid, but other acids can alsobe used, such as acetic acid, citric acid, nitric acid, phosphoric acid,tartaric acid, succinic acid, hydrogen chloride, or mixtures thereof.Mild acid treatment is conducted in the pH range of preferably 1-5,e.g., 1-4 or 1-2.5. In one aspect, the acid concentration is in therange from preferably 0.01 to 10 wt % acid, e.g., 0.05 to 5 wt % acid or0.1 to 2 wt % acid. The acid is contacted with the cellulosic materialand held at a temperature in the range of preferably 140-200° C., e.g.,165-190° C., for periods ranging from 1 to 60 minutes.

In another aspect, pretreatment takes place in an aqueous slurry. Inpreferred aspects, the cellulosic material is present duringpretreatment in amounts preferably between 10-80 wt %, e.g., 20-70 wt %or 30-60 wt %, such as around 40 wt %. The pretreated cellulosicmaterial can be unwashed or washed using any method known in the art,e.g., washed with water.

Mechanical Pretreatment or Physical Pretreatment: The term “mechanicalpretreatment” or “physical pretreatment” refers to any pretreatment thatpromotes size reduction of particles. For example, such pretreatment caninvolve various types of grinding or milling (e.g., dry milling, wetmilling, or vibratory ball milling).

The cellulosic material can be pretreated both physically (mechanically)and chemically. Mechanical or physical pretreatment can be coupled withsteaming/steam explosion, hydrothermolysis, dilute or mild acidtreatment, high temperature, high pressure treatment, irradiation (e.g.,microwave irradiation), or combinations thereof. In one aspect, highpressure means pressure in the range of preferably about 100 to about400 psi, e.g., about 150 to about 250 psi. In another aspect, hightemperature means temperatures in the range of about 100 to about 300°C., e.g., about 140 to about 200° C. In a preferred aspect, mechanicalor physical pretreatment is performed in a batch-process using a steamgun hydrolyzer system that uses high pressure and high temperature asdefined above, e.g., a Sunds Hydrolyzer available from Sunds DefibratorAB, Sweden. The physical and chemical pretreatments can be carried outsequentially or simultaneously, as desired.

Accordingly, in a preferred aspect, the cellulosic material is subjectedto physical (mechanical) or chemical pretreatment, or any combinationthereof, to promote the separation and/or release of cellulose,hemicellulose, and/or lignin.

Biological Pretreatment: The term “biological pretreatment” refers toany biological pretreatment that promotes the separation and/or releaseof cellulose, hemicellulose, and/or lignin from the cellulosic material.Biological pretreatment techniques can involve applyinglignin-solubilizing microorganisms and/or enzymes (see, for example,Hsu, T.-A., 1996, Pretreatment of biomass, in Handbook on Bioethanol:Production and Utilization, Wyman, C. E., ed., Taylor & Francis,Washington, D.C., 179-212; Ghosh and Singh, 1993, Physicochemical andbiological treatments for enzymatic/microbial conversion of cellulosicbiomass, Adv. Appl. Microbiol. 39: 295-333; McMillan, J. D., 1994,Pretreating lignocellulosic biomass: a review, in Enzymatic Conversionof Biomass for Fuels Production, Himmel, M. E., Baker, J. O., andOverend, R. P., eds., ACS Symposium Series 566, American ChemicalSociety, Washington, D.C., chapter 15; Gong, C. S., Cao, N. J., Du, J.,and Tsao, G. T., 1999, Ethanol production from renewable resources, inAdvances in Biochemical Engineering/Biotechnology, Scheper, T., ed.,Springer-Verlag Berlin Heidelberg, Germany, 65: 207-241; Olsson andHahn-Hagerdal, 1996, Fermentation of lignocellulosic hydrolysates forethanol production, Enz. Microb. Tech. 18: 312-331; and Vallander andEriksson, 1990, Production of ethanol from lignocellulosic materials:State of the art, Adv. Biochem. Eng./Biotechnol. 42: 63-95).

Saccharification.

In the hydrolysis step, also known as saccharification, the cellulosicmaterial, e.g., pretreated, is hydrolyzed to break down cellulose and/orhemicellulose to fermentable sugars, such as glucose, cellobiose,xylose, xylulose, arabinose, mannose, galactose, and/or solubleoligosaccharides. The hydrolysis is performed enzymatically by an enzymecomposition as described herein in the presence of a polypeptide havingcellulolytic enhancing activity of the present invention. The enzymes ofthe compositions can be added simultaneously or sequentially.

Enzymatic hydrolysis is preferably carried out in a suitable aqueousenvironment under conditions that can be readily determined by oneskilled in the art. In one aspect, hydrolysis is performed underconditions suitable for the activity of the enzyme(s), i.e., optimal forthe enzyme(s). The hydrolysis can be carried out as a fed batch orcontinuous process where the cellulosic material is fed gradually to,for example, an enzyme containing hydrolysis solution.

The saccharification is generally performed in stirred-tank reactors orfermentors under controlled pH, temperature, and mixing conditions.Suitable process time, temperature and pH conditions can readily bedetermined by one skilled in the art. For example, the saccharificationcan last up to 200 hours, but is typically performed for preferablyabout 12 to about 120 hours, e.g., about 16 to about 72 hours or about24 to about 48 hours. The temperature is in the range of preferablyabout 25° C. to about 70° C., e.g., about 30° C. to about 65° C., about40° C. to about 60° C., or about 50° C. to about 55° C. The pH is in therange of preferably about 3 to about 8, e.g., about 3.5 to about 7,about 4 to about 6, or about 5.0 to about 5.5. The dry solids content isin the range of preferably about 5 to about 50 wt %, e.g., about 10 toabout 40 wt % or about 20 to about 30 wt %.

The enzyme compositions can comprise any protein useful in degrading thecellulosic material.

In one aspect, the enzyme composition comprises or further comprises oneor more (e.g., several) proteins selected from the group consisting of acellulase, a GH61 polypeptide having cellulolytic enhancing activity, ahemicellulase, an esterase, an expansin, a laccase, a ligninolyticenzyme, a pectinase, a peroxidase, a protease, and a swollenin. Inanother aspect, the cellulase is preferably one or more (e.g., several)enzymes selected from the group consisting of an endoglucanase, acellobiohydrolase, and a beta-glucosidase. In another aspect, thehemicellulase is preferably one or more (e.g., several) enzymes selectedfrom the group consisting of an acetylmannan esterase, an acetylxylanesterase, an arabinanase, an arabinofuranosidase, a coumaric acidesterase, a feruloyl esterase, a galactosidase, a glucuronidase, aglucuronoyl esterase, a mannanase, a mannosidase, a xylanase, and axylosidase.

In another aspect, the enzyme composition comprises one or more (e.g.,several) cellulolytic enzymes. In another aspect, the enzyme compositioncomprises or further comprises one or more (e.g., several)hemicellulolytic enzymes. In another aspect, the enzyme compositioncomprises one or more (e.g., several) cellulolytic enzymes and one ormore (e.g., several) hemicellulolytic enzymes. In another aspect, theenzyme composition comprises one or more (e.g., several) enzymesselected from the group of cellulolytic enzymes and hemicellulolyticenzymes. In another aspect, the enzyme composition comprises anendoglucanase. In another aspect, the enzyme composition comprises acellobiohydrolase. In another aspect, the enzyme composition comprises abeta-glucosidase. In another aspect, the enzyme composition comprises apolypeptide having cellulolytic enhancing activity. In another aspect,the enzyme composition comprises an endoglucanase and a polypeptidehaving cellulolytic enhancing activity. In another aspect, the enzymecomposition comprises a cellobiohydrolase and a polypeptide havingcellulolytic enhancing activity. In another aspect, the enzymecomposition comprises a beta-glucosidase and a polypeptide havingcellulolytic enhancing activity. In another aspect, the enzymecomposition comprises an endoglucanase and a cellobiohydrolase. Inanother aspect, the enzyme composition comprises an endoglucanase and abeta-glucosidase. In another aspect, the enzyme composition comprises acellobiohydrolase and a beta-glucosidase. In another aspect, the enzymecomposition comprises an endoglucanase, a cellobiohydrolase, and apolypeptide having cellulolytic enhancing activity. In another aspect,the enzyme composition comprises an endoglucanase, a beta-glucosidase,and a polypeptide having cellulolytic enhancing activity. In anotheraspect, the enzyme composition comprises a cellobiohydrolase, abeta-glucosidase, and a polypeptide having cellulolytic enhancingactivity. In another aspect, the enzyme composition comprises anendoglucanase, a cellobiohydrolase, and a beta-glucosidase. In anotheraspect, the enzyme composition comprises an endoglucanase, acellobiohydrolase, a beta-glucosidase, and a polypeptide havingcellulolytic enhancing activity.

In another aspect, the enzyme composition comprises an acetylmannanesterase. In another aspect, the enzyme composition comprises anacetylxylan esterase. In another aspect, the enzyme compositioncomprises an arabinanase (e.g., alpha-L-arabinanase). In another aspect,the enzyme composition comprises an arabinofuranosidase (e.g.,alpha-L-arabinofuranosidase). In another aspect, the enzyme compositioncomprises a coumaric acid esterase. In another aspect, the enzymecomposition comprises a feruloyl esterase. In another aspect, the enzymecomposition comprises a galactosidase (e.g., alpha-galactosidase and/orbeta-galactosidase). In another aspect, the enzyme composition comprisesa glucuronidase (e.g., alpha-D-glucuronidase). In another aspect, theenzyme composition comprises a glucuronoyl esterase. In another aspect,the enzyme composition comprises a mannanase. In another aspect, theenzyme composition comprises a mannosidase (e.g., beta-mannosidase). Inanother aspect, the enzyme composition comprises a xylanase. In apreferred aspect, the xylanase is a Family 10 xylanase. In anotheraspect, the enzyme composition comprises a xylosidase (e.g.,beta-xylosidase).

In another aspect, the enzyme composition comprises an esterase. Inanother aspect, the enzyme composition comprises an expansin. In anotheraspect, the enzyme composition comprises a laccase. In another aspect,the enzyme composition comprises a ligninolytic enzyme. In a preferredaspect, the ligninolytic enzyme is a manganese peroxidase. In anotherpreferred aspect, the ligninolytic enzyme is a lignin peroxidase. Inanother preferred aspect, the ligninolytic enzyme is a H₂O₂-producingenzyme. In another aspect, the enzyme composition comprises a pectinase.In another aspect, the enzyme composition comprises a peroxidase. Inanother aspect, the enzyme composition comprises a protease. In anotheraspect, the enzyme composition comprises a swollenin.

In the processes of the present invention, the enzyme(s) can be addedprior to or during fermentation, e.g., during saccharification or duringor after propagation of the fermenting microorganism(s).

One or more (e.g., several) components of the enzyme composition may bewild-type proteins, recombinant proteins, or a combination of wild-typeproteins and recombinant proteins. For example, one or more (e.g.,several) components may be native proteins of a cell, which is used as ahost cell to express recombinantly one or more (e.g., several) othercomponents of the enzyme composition. One or more (e.g., several)components of the enzyme composition may be produced as monocomponents,which are then combined to form the enzyme composition. The enzymecomposition may be a combination of multicomponent and monocomponentprotein preparations.

The enzymes used in the processes of the present invention may be in anyform suitable for use, such as, for example, a fermentation brothformulation or a cell composition, a cell lysate with or withoutcellular debris, a semi-purified or purified enzyme preparation, or ahost cell as a source of the enzymes. The enzyme composition may be adry powder or granulate, a non-dusting granulate, a liquid, a stabilizedliquid, or a stabilized protected enzyme. Liquid enzyme preparationsmay, for instance, be stabilized by adding stabilizers such as a sugar,a sugar alcohol or another polyol, and/or lactic acid or another organicacid according to established processes.

The optimum amounts of the enzymes and a polypeptide having cellulolyticenhancing activity depend on several factors including, but not limitedto, the mixture of component cellulolytic enzymes, the cellulosicmaterial, the concentration of cellulosic material, the pretreatment(s)of the cellulosic material, temperature, time, pH, and inclusion offermenting organism (e.g., yeast for Simultaneous Saccharification andFermentation).

In one aspect, an effective amount of cellulolytic or hemicellulolyticenzyme to the cellulosic material is about 0.5 to about 50 mg, e.g.,about 0.5 to about 40 mg, about 0.5 to about 25 mg, about 0.75 to about20 mg, about 0.75 to about 15 mg, about 0.5 to about 10 mg, or about 2.5to about 10 mg per g of the cellulosic material.

In another aspect, an effective amount of a polypeptide havingcellulolytic enhancing activity to the cellulosic material is about 0.01to about 50.0 mg, e.g., about 0.01 to about 40 mg, about 0.01 to about30 mg, about 0.01 to about 20 mg, about 0.01 to about 10 mg, about 0.01to about 5 mg, about 0.025 to about 1.5 mg, about 0.05 to about 1.25 mg,about 0.075 to about 1.25 mg, about 0.1 to about 1.25 mg, about 0.15 toabout 1.25 mg, or about 0.25 to about 1.0 mg per g of the cellulosicmaterial.

In another aspect, an effective amount of a polypeptide havingcellulolytic enhancing activity to cellulolytic or hemicellulolyticenzyme is about 0.005 to about 1.0 g, e.g., about 0.01 to about 1.0 g,about 0.15 to about 0.75 g, about 0.15 to about 0.5 g, about 0.1 toabout 0.5 g, about 0.1 to about 0.25 g, or about 0.05 to about 0.2 g perg of cellulolytic or hemicellulolytic enzyme.

The polypeptides having cellulolytic enzyme activity or hemicellulolyticenzyme activity as well as other proteins/polypeptides useful in thedegradation of the cellulosic material, e.g., GH61 polypeptides havingcellulolytic enhancing activity (collectively hereinafter “polypeptideshaving enzyme activity”) can be derived or obtained from any suitableorigin, including, bacterial, fungal, yeast, plant, or mammalian origin.The term “obtained” also means herein that the enzyme may have beenproduced recombinantly in a host organism employing methods describedherein, wherein the recombinantly produced enzyme is either native orforeign to the host organism or has a modified amino acid sequence,e.g., having one or more (e.g., several) amino acids that are deleted,inserted and/or substituted, i.e., a recombinantly produced enzyme thatis a mutant and/or a fragment of a native amino acid sequence or anenzyme produced by nucleic acid shuffling processes known in the art.Encompassed within the meaning of a native enzyme are natural variantsand within the meaning of a foreign enzyme are variants obtainedrecombinantly, such as by site-directed mutagenesis or shuffling.

A polypeptide having enzyme activity may be a bacterial polypeptide. Forexample, the polypeptide may be a gram positive bacterial polypeptidesuch as a Bacillus, Streptococcus, Streptomyces, Staphylococcus,Enterococcus, Lactobacillus, Lactococcus, Clostridium, Geobacillus,Caldicellulosiruptor, Acidothermus, Thermobifidia, or Oceanobacilluspolypeptide having enzyme activity, or a Gram negative bacterialpolypeptide such as an E. coli, Pseudomonas, Salmonella, Campylobacter,Helicobacter, Flavobacterium, Fusobacterium, Ilyobacter, Neisseria, orUreaplasma polypeptide having enzyme activity.

In one aspect, the polypeptide is a Bacillus alkalophilus, Bacillusamyloliquefaciens, Bacillus brevis, Bacillus circulans, Bacillusclausii, Bacillus coagulans, Bacillus firmus, Bacillus lautus, Bacilluslentus, Bacillus licheniformis, Bacillus megaterium, Bacillus pumilus,Bacillus stearothermophilus, Bacillus subtilis, or Bacillusthuringiensis polypeptide having enzyme activity.

In another aspect, the polypeptide is a Streptococcus equisimilis,Streptococcus pyogenes, Streptococcus uberis, or Streptococcus equisubsp. Zooepidemicus polypeptide having enzyme activity.

In another aspect, the polypeptide is a Streptomyces achromogenes,Streptomyces avermitilis, Streptomyces coelicolor, Streptomyces griseus,or Streptomyces lividans polypeptide having enzyme activity.

The polypeptide having enzyme activity may also be a fungal polypeptide,and more preferably a yeast polypeptide such as a Candida,Kluyveromyces, Pichia, Saccharomyces, Schizosaccharomyces, or Yarrowiapolypeptide having enzyme activity; or more preferably a filamentousfungal polypeptide such as an Acremonium, Agaricus, Alternaria,Aspergillus, Aureobasidium, Botryospaeria, Ceriporiopsis, Chaetomidium,Chrysosporium, Claviceps, Cochliobolus, Coprinopsis, Coptotermes,Corynascus, Cryphonectria, Cryptococcus, Diplodia, Exidia, Filibasidium,Fusarium, Gibberella, Holomastigotoides, Humicola, Irpex, Lentinula,Leptospaeria, Magnaporthe, Melanocarpus, Meripilus, Mucor,Myceliophthora, Neocallimastix, Neurospora, Paecilomyces, Penicillium,Phanerochaete, Piromyces, Poitrasia, Pseudoplectania,Pseudotrichonympha, Rhizomucor, Schizophyllum, Scytalidium, Talaromyces,Thermoascus, Thielavia, Tolypocladium, Trichoderma, Trichophaea,Verticillium, Volvariella, or Xylaria polypeptide having enzymeactivity.

In one aspect, the polypeptide is a Saccharomyces carlsbergensis,Saccharomyces cerevisiae, Saccharomyces diastaticus, Saccharomycesdouglasii, Saccharomyces kluyveri, Saccharomyces norbensis, orSaccharomyces oviformis polypeptide having enzyme activity.

In another aspect, the polypeptide is an Acremonium cellulolyticus,Aspergillus aculeatus, Aspergillus awamori, Aspergillus fumigatus,Aspergillus foetidus, Aspergillus japonicus, Aspergillus nidulans,Aspergillus niger, Aspergillus oryzae, Chrysosporium keratinophilum,Chrysosporium lucknowense, Chrysosporium tropicum, Chrysosporiummerdarium, Chrysosporium inops, Chrysosporium pannicola, Chrysosporiumqueenslandicum, Chrysosporium zonatum, Fusarium bactridioides, Fusariumcerealis, Fusarium crookwellense, Fusarium culmorum, Fusariumgraminearum, Fusarium graminum, Fusarium heterosporum, Fusarium negundi,Fusarium oxysporum, Fusarium reticulatum, Fusarium roseum, Fusariumsambucinum, Fusarium sarcochroum, Fusarium sporotrichioides, Fusariumsulphureum, Fusarium torulosum, Fusarium trichothecioides, Fusariumvenenatum, Humicola grisea, Humicola insolens, Humicola lanuginosa,Irpex lacteus, Mucor miehei, Myceliophthora thermophila, Neurosporacrassa, Penicillium funiculosum, Penicillium purpurogenum, Phanerochaetechrysosporium, Thielavia achromatica, Thielavia albomyces, Thielaviaalbopilosa, Thielavia australeinsis, Thielavia fimeti, Thielaviamicrospora, Thielavia ovispora, Thielavia peruviana, Thielaviaspededonium, Thielavia setosa, Thielavia subthermophila, Thielaviaterrestris, Trichoderma harzianum, Trichoderma koningii, Trichodermalongibrachiatum, Trichoderma reesei, Trichoderma viride, or Trichophaeasaccata polypeptide having enzyme activity.

Chemically modified or protein engineered mutants of polypeptides havingenzyme activity may also be used.

One or more (e.g., several) components of the enzyme composition may bea recombinant component, i.e., produced by cloning of a DNA sequenceencoding the single component and subsequent cell transformed with theDNA sequence and expressed in a host (see, for example, WO 91/17243 andWO 91/17244). The host is preferably a heterologous host (enzyme isforeign to host), but the host may under certain conditions also be ahomologous host (enzyme is native to host). Monocomponent cellulolyticproteins may also be prepared by purifying such a protein from afermentation broth.

In one aspect, the one or more (e.g., several) cellulolytic enzymescomprise a commercial cellulolytic enzyme preparation. Examples ofcommercial cellulolytic enzyme preparations suitable for use in thepresent invention include, for example, CELLIC® CTec (Novozymes NS),CELLIC® CTec2 (Novozymes NS), CELLUCLAST™ (Novozymes NS), NOVOZYM™ 188(Novozymes NS), CELLUZYME™ (Novozymes NS), CEREFLO™ (Novozymes NS), andULTRAFLO™ (Novozymes NS), ACCELERASE™ (Genencor Int.), LAMINEX™(Genencor Int.), SPEZYME™ CP (Genencor Int.), FILTRASE® NL (DSM);METHAPLUS® S/L 100 (DSM), ROHAMENT™ 7069 W (Röhm GmbH), FIBREZYME® LDI(Dyadic International, Inc.), FIBREZYME® LBR (Dyadic International,Inc.), or VISCOSTAR® 150L (Dyadic International, Inc.). The cellulaseenzymes are added in amounts effective from about 0.001 to about 5.0 wt% of solids, e.g., about 0.025 to about 4.0 wt % of solids or about0.005 to about 2.0 wt % of solids.

Examples of bacterial endoglucanases that can be used in the processesof the present invention, include, but are not limited to, anAcidothermus cellulolyticus endoglucanase (WO 91/05039; WO 93/15186;U.S. Pat. No. 5,275,944; WO 96/02551; U.S. Pat. No. 5,536,655, WO00/70031, WO 05/093050); Thermobifida fusca endoglucanase III (WO05/093050); and Thermobifida fusca endoglucanase V (WO 05/093050).

Examples of fungal endoglucanases that can be used in the presentinvention, include, but are not limited to, a Trichoderma reeseiendoglucanase I (Penttila et al., 1986, Gene 45: 253-263, Trichodermareesei Cel7B endoglucanase I (GENBANK™ accession no. M15665),Trichoderma reesei endoglucanase II (Saloheimo, et al., 1988, Gene63:11-22), Trichoderma reesei Cel5A endoglucanase II (GENBANK™ accessionno. M19373), Trichoderma reesei endoglucanase III (Okada et al., 1988,Appl. Environ. Microbiol. 64: 555-563, GENBANK™ accession no. AB003694),Trichoderma reesei endoglucanase V (Saloheimo et al., 1994, MolecularMicrobiology 13: 219-228, GENBANK™ accession no. Z33381), Aspergillusaculeatus endoglucanase (Ooi et al., 1990, Nucleic Acids Research 18:5884), Aspergillus kawachii endoglucanase (Sakamoto et al., 1995,Current Genetics 27: 435-439), Erwinia carotovara endoglucanase(Saarilahti et al., 1990, Gene 90: 9-14), Fusarium oxysporumendoglucanase (GENBANK™ accession no. L29381), Humicola grisea var.thermoidea endoglucanase (GENBANK™ accession no. AB003107), Melanocarpusalbomyces endoglucanase (GENBANK™ accession no. MAL515703), Neurosporacrassa endoglucanase (GENBANK™ accession no. XM_324477), Humicolainsolens endoglucanase V, Myceliophthora thermophila CBS 117.65endoglucanase, basidiomycete CBS 495.95 endoglucanase, basidiomycete CBS494.95 endoglucanase, Thielavia terrestris NRRL 8126 CEL6Bendoglucanase, Thielavia terrestris NRRL 8126 CEL6C endoglucanase,Thielavia terrestris NRRL 8126 CEL7C endoglucanase, Thielavia terrestrisNRRL 8126 CEL7E endoglucanase, Thielavia terrestris NRRL 8126 CEL7Fendoglucanase, Cladorrhinum foecundissimum ATCC 62373 CEL7Aendoglucanase, and Trichoderma reesei strain No. VTT-D-80133endoglucanase (GENBANK™ accession no. M15665).

Examples of cellobiohydrolases useful in the present invention include,but are not limited to, Aspergillus aculeatus cellobiohydrolase II (WO2011/059740), Chaetomium thermophilum cellobiohydrolase I, Chaetomiumthermophilum cellobiohydrolase II, Humicola insolens cellobiohydrolaseI, Myceliophthora thermophila cellobiohydrolase II (WO 2009/042871),Thielavia hyrcanie cellobiohydrolase II (WO 2010/141325), Thielaviaterrestris cellobiohydrolase II (CEL6A, WO 2006/074435), Trichodermareesei cellobiohydrolase I, Trichoderma reesei cellobiohydrolase II, andTrichophaea saccata cellobiohydrolase II (WO 2010/057086).

Examples of beta-glucosidases useful in the present invention include,but are not limited to, beta-glucosidases from Aspergillus aculeatus(Kawaguchi et al., 1996, Gene 173: 287-288), Aspergillus fumigatus (WO2005/047499), Aspergillus niger (Dan et al., 2000, J. Biol. Chem. 275:4973-4980), Aspergillus oryzae (WO 2002/095014), Penicillium brasilianumIBT 20888 (WO 2007/019442 and WO 2010/088387), Thielavia terrestris (WO2011/035029), and Trichophaea saccata (WO 2007/019442).

The beta-glucosidase may be a fusion protein. In one aspect, thebeta-glucosidase is an Aspergillus oryzae beta-glucosidase variant BGfusion protein (WO 2008/057637) or an Aspergillus oryzaebeta-glucosidase fusion protein (WO 2008/057637).

Other useful endoglucanases, cellobiohydrolases, and beta-glucosidasesare disclosed in numerous Glycosyl Hydrolase families using theclassification according to Henrissat B., 1991, A classification ofglycosyl hydrolases based on amino-acid sequence similarities, Biochem.J. 280: 309-316, and Henrissat B., and Bairoch A., 1996, Updating thesequence-based classification of glycosyl hydrolases, Biochem. J. 316:695-696.

Other cellulolytic enzymes that may be used in the present invention aredescribed in WO 98/13465, WO 98/015619, WO 98/015633, WO 99/06574, WO99/10481, WO 99/025847, WO 99/031255, WO 2002/101078, WO 2003/027306, WO2003/052054, WO 2003/052055, WO 2003/052056, WO 2003/052057, WO2003/052118, WO 2004/016760, WO 2004/043980, WO 2004/048592, WO2005/001065, WO 2005/028636, WO 2005/093050, WO 2005/093073, WO2006/074005, WO 2006/117432, WO 2007/071818, WO 2007/071820, WO2008/008070, WO 2008/008793, U.S. Pat. No. 5,457,046, U.S. Pat. No.5,648,263, and U.S. Pat. No. 5,686,593.

In one aspect, the GH61 polypeptide having cellulolytic enhancingactivity is used in the presence of a soluble activating divalent metalcation according to WO 2008/151043, e.g., manganese sulfate.

In one aspect, the GH61 polypeptide having cellulolytic enhancingactivity is used in the presence of a dioxy compound, a bicyliccompound, a heterocyclic compound, a nitrogen-containing compound, aquinone compound, a sulfur-containing compound, or a liquor obtainedfrom a pretreated cellulosic material such as pretreated corn stover(PCS).

The dioxy compound may include any suitable compound containing two ormore oxygen atoms. In some aspects, the dioxy compounds contain asubstituted aryl moiety as described herein. The dioxy compounds maycomprise one or more (e.g., several) hydroxyl and/or hydroxylderivatives, but also include substituted aryl moieties lacking hydroxyland hydroxyl derivatives. Non-limiting examples of the dioxy compoundsinclude pyrocatechol or catechol; caffeic acid; 3,4-dihydroxybenzoicacid; 4-tert-butyl-5-methoxy-1,2-benzenediol; pyrogallol; gallic acid;methyl-3,4,5-trihydroxybenzoate; 2,3,4-trihydroxybenzophenone;2,6-dimethoxyphenol; sinapinic acid; 3,5-dihydroxybenzoic acid;4-chloro-1,2-benzenediol; 4-nitro-1,2-benzenediol; tannic acid; ethylgallate; methyl glycolate; dihydroxyfumaric acid; 2-butyne-1,4-diol;(croconic acid; 1,3-propanediol; tartaric acid; 2,4-pentanediol;3-ethyoxy-1,2-propanediol; 2,4,4′-trihydroxybenzophenone;cis-2-butene-1,4-diol; 3,4-dihydroxy-3-cyclobutene-1,2-dione;dihydroxyacetone; acrolein acetal; methyl-4-hydroxybenzoate;4-hydroxybenzoic acid; and methyl-3,5-dimethoxy-4-hydroxybenzoate; or asalt or solvate thereof.

The bicyclic compound may include any suitable substituted fused ringsystem as described herein. The compounds may comprise one or more(e.g., several) additional rings, and are not limited to a specificnumber of rings unless otherwise stated. In one aspect, the bicycliccompound is a flavonoid. In another aspect, the bicyclic compound is anoptionally substituted isoflavonoid. In another aspect, the bicycliccompound is an optionally substituted flavylium ion, such as anoptionally substituted anthocyanidin or optionally substitutedanthocyanin, or derivative thereof. Non-limiting examples of thebicycliccompounds include epicatechin; quercetin; myricetin; taxifolin;kaempferol; morin; acacetin; naringenin; isorhamnetin; apigenin;cyanidin; cyanin; kuromanin; keracyanin; or a salt or solvate thereof.

The heterocyclic compound may be any suitable compound, such as anoptionally substituted aromatic or non-aromatic ring comprising aheteroatom, as described herein. In one aspect, the heterocyclic is acompound comprising an optionally substituted heterocycloalkyl moiety oran optionally substituted heteroaryl moiety. In another aspect, theoptionally substituted heterocycloalkyl moiety or optionally substitutedheteroaryl moiety is an optionally substituted 5-memberedheterocycloalkyl or an optionally substituted 5-membered heteroarylmoiety. In another aspect, the optionally substituted heterocycloalkylor optionally substituted heteroaryl moiety is an optionally substitutedmoiety selected from pyrazolyl, furanyl, imidazolyl, isoxazolyl,oxadiazolyl, oxazolyl, pyrrolyl, pyridyl, pyrimidyl, pyridazinyl,thiazolyl, triazolyl, thienyl, dihydrothieno-pyrazolyl, thianaphthenyl,carbazolyl, benzimidazolyl, benzothienyl, benzofuranyl, indolyl,quinolinyl, benzotriazolyl, benzothiazolyl, benzooxazolyl,benzimidazolyl, isoquinolinyl, isoindolyl, acridinyl, benzoisazolyl,dimethylhydantoin, pyrazinyl, tetrahydrofuranyl, pyrrolinyl,pyrrolidinyl, morpholinyl, indolyl, diazepinyl, azepinyl, thiepinyl,piperidinyl, and oxepinyl. In another aspect, the optionally substitutedheterocycloalkyl moiety or optionally substituted heteroaryl moiety isan optionally substituted furanyl. Non-limiting examples of theheterocyclic compounds include(1,2-dihydroxyethyl)-3,4-dihydroxyfuran-2(5H)-one;4-hydroxy-5-methyl-3-furanone; 5-hydroxy-2(5H)-furanone;[1,2-dihydroxyethyl]furan-2,3,4(5H)-trione; α-hydroxy-γ-butyrolactone;ribonic γ-lactone; aldohexuronicaldohexuronic acid γ-lactone; gluconicacid 5-lactone; 4-hydroxycoumarin; dihydrobenzofuran;5-(hydroxymethyl)furfural; furoin; 2(5H)-furanone;5,6-dihydro-2H-pyran-2-one; and5,6-dihydro-4-hydroxy-6-methyl-2H-pyran-2-one; or a salt or solvatethereof.

The nitrogen-containing compound may be any suitable compound with oneor more nitrogen atoms. In one aspect, the nitrogen-containing compoundcomprises an amine, imine, hydroxylamine, or nitroxide moiety.Non-limiting examples of thenitrogen-containing compounds includeacetone oxime; violuric acid; pyridine-2-aldoxime; 2-aminophenol;1,2-benzenediamine; 2,2,6,6-tetramethyl-1-piperidinyloxy;5,6,7,8-tetrahydrobiopterin; 6,7-dimethyl-5,6,7,8-tetrahydropterine; andmaleamic acid; or a salt or solvate thereof.

The quinone compound may be any suitable compound comprising a quinonemoiety as described herein. Non-limiting examples of the quinonecompounds include 1,4-benzoquinone; 1,4-naphthoquinone;2-hydroxy-1,4-naphthoquinone; 2,3-dimethoxy-5-methyl-1,4-benzoquinone orcoenzyme Q₀; 2,3,5,6-tetramethyl-1,4-benzoquinone or duroquinone;1,4-dihydroxyanthraquinone; 3-hydroxy-1-methyl-5,6-indolinedione oradrenochrome; 4-tert-butyl-5-methoxy-1,2-benzoquinone; pyrroloquinolinequinone; or a salt or solvate thereof.

The sulfur-containing compound may be any suitable compound comprisingone or more sulfur atoms. In one aspect, the sulfur-containing comprisesa moiety selected from thionyl, thioether, sulfinyl, sulfonyl,sulfamide, sulfonamide, sulfonic acid, and sulfonic ester. Non-limitingexamples of the sulfur-containing compounds include ethanethiol;2-propanethiol; 2-propene-1-thiol; 2-mercaptoethanesulfonic acid;benzenethiol; benzene-1,2-dithiol; cysteine; methionine; glutathione;cystine; or a salt or solvate thereof.

In one aspect, an effective amount of such a compound described above tocellulosic material as a molar ratio to glucosyl units of cellulose isabout 10⁻⁶ to about 10, e.g., about 10⁻⁶ to about 7.5, about 10⁻⁶ toabout 5, about 10⁻⁶ to about 2.5, about 10⁻⁶ to about 1, about 10⁻⁶ toabout 1, about 10⁻⁶ to about 10⁻¹, about 10⁻⁴ to about 10⁻¹, about 10⁻³to about 10⁻¹, or about 10⁻³ to about 10⁻². In another aspect, aneffective amount of such a compound described above is about 0.1 μM toabout 1 M, e.g., about 0.5 μM to about 0.75 M, about 0.75 μM to about0.5 M, about 1 μM to about 0.25 M, about 1 μM to about 0.1 M, about 5 μMto about 50 mM, about 10 μM to about 25 mM, about 50 μM to about 25 mM,about 10 μM to about 10 mM, about 5 μM to about 5 mM, or about 0.1 mM toabout 1 mM.

The term “liquor” means the solution phase, either aqueous, organic, ora combination thereof, arising from treatment of a lignocellulose and/orhemicellulose material in a slurry, or monosaccharides thereof, e.g.,xylose, arabinose, mannose, etc., under conditions as described herein,and the soluble contents thereof. A liquor for cellulolytic enhancementof a GH61 polypeptide can be produced by treating a lignocellulose orhemicellulose material (or feedstock) by applying heat and/or pressure,optionally in the presence of a catalyst, e.g., acid, optionally in thepresence of an organic solvent, and optionally in combination withphysical disruption of the material, and then separating the solutionfrom the residual solids. Such conditions determine the degree ofcellulolytic enhancement obtainable through the combination of liquorand a GH61 polypeptide during hydrolysis of a cellulosic substrate by acellulase preparation. The liquor can be separated from the treatedmaterial using a method standard in the art, such as filtration,sedimentation, or centrifugation.

In one aspect, an effective amount of the liquor to cellulose is about10⁻⁶ to about 10 g per g of cellulose, e.g., about 10⁻⁶ to about 7.5 g,about 10⁻⁶ to about 5, about 10⁻⁶ to about 2.5 g, about 10⁻⁶ to about 1g, about 10⁻⁵ to about 1 g, about 10⁻⁵ to about 10⁻¹ g, about 10⁻⁴ toabout 10⁻¹ g, about 10⁻³ to about 10⁻¹ g, or about 10⁻³ to about 10⁻² gper g of cellulose.

In one aspect, the one or more (e.g., several) hemicellulolytic enzymescomprise a commercial hemicellulolytic enzyme preparation. Examples ofcommercial hemicellulolytic enzyme preparations suitable for use in thepresent invention include, for example, SHEARZYME™ (Novozymes NS),CELLIC® HTec (Novozymes NS), CELLIC® HTec2 (Novozymes NS), VISCOZYME®(Novozymes NS), ULTRAFLO® (Novozymes NS), PULPZYME® HC (Novozymes NS),MULTIFECT® Xylanase (Genencor), ACCELLERASE® XY (Genencor), ACCELLERASE®XC (Genencor), ECOPULP® TX-200A (AB Enzymes), HSP 6000 Xylanase (DSM),DEPOL™ 333P (Biocatalysts Limit, Wales, UK), DEPOL™ 740L. (BiocatalystsLimit, Wales, UK), and DEPOL™ 762P (Biocatalysts Limit, Wales, UK).

Examples of xylanases useful in the processes of the present inventioninclude, but are not limited to, xylanases from Aspergillus aculeatus(GeneSeqP:AAR63790; WO 94/21785), Aspergillus fumigatus (WO2006/078256), Penicillium pinophilum (WO 2011/041405), Penicillium sp.(WO 2010/126772), Thielavia terrestris NRRL 8126 (WO 2009/079210), andTrichophaea saccata GH10 (WO 2011/057083).

Examples of beta-xylosidases useful in the processes of the presentinvention include, but are not limited to, beta-xylosidases fromNeurospora crassa (SwissProt accession number Q7SOW4), Trichodermareesei (UniProtKB/TrEMBL accession number Q92458), and Talaromycesemersonii (SwissProt accession number Q8X212).

Examples of acetylxylan esterases useful in the processes of the presentinvention include, but are not limited to, acetylxylan esterases fromAspergillus aculeatus (WO 2010/108918), Chaetomium globosum (Uniprotaccession number Q2GWX4), Chaetomium gracile (GeneSeqP accession numberAAB82124), Humicola insolens DSM 1800 (WO 2009/073709), Hypocreajecorina (WO 2005/001036), Myceliophtera thermophila (WO 2010/014880),Neurospora crassa (UniProt accession number q7s259), Phaeosphaerianodorum (Uniprot accession number Q0UHJ1), and Thielavia terrestris NRRL8126 (WO 2009/042846).

Examples of feruloyl esterases (ferulic acid esterases) useful in theprocesses of the present invention include, but are not limited to,feruloyl esterases form Humicola insolens DSM 1800 (WO 2009/076122),Neosartorya fischeri (UniProt Accession number A1D9T4), Neurosporacrassa (UniProt accession number Q9HGR3), Penicillium aurantiogriseum(WO 2009/127729), and Thielavia terrestris (WO 2010/053838 and WO2010/065448).

Examples of arabinofuranosidases useful in the processes of the presentinvention include, but are not limited to, arabinofuranosidases fromAspergillus niger (GeneSeqP accession number AAR94170), Humicolainsolens DSM 1800 (WO 2006/114094 and WO 2009/073383), and M. giganteus(WO 2006/114094).

Examples of alpha-glucuronidases useful in the processes of the presentinvention include, but are not limited to, alpha-glucuronidases fromAspergillus clavatus (UniProt accession number alcc12), Aspergillusfumigatus (SwissProt accession number Q4WW45), Aspergillus niger(Uniprot accession number Q96WX9), Aspergillus terreus (SwissProtaccession number Q0CJP9), Humicola insolens (WO 2010/014706),Penicillium aurantiogriseum (WO 2009/068565), Talaromyces emersonii(UniProt accession number Q8X211), and Trichoderma reesei (Uniprotaccession number Q99024).

The polypeptides having enzyme activity used in the processes of thepresent invention may be produced by fermentation of the above-notedmicrobial strains on a nutrient medium containing suitable carbon andnitrogen sources and inorganic salts, using procedures known in the art(see, e.g., Bennett, J. W. and LaSure, L. (eds.), More GeneManipulations in Fungi, Academic Press, CA, 1991). Suitable media areavailable from commercial suppliers or may be prepared according topublished compositions (e.g., in catalogues of the American Type CultureCollection). Temperature ranges and other conditions suitable for growthand enzyme production are known in the art (see, e.g., Bailey, J. E.,and Ollis is, D. F., Biochemical Engineering Fundamentals, McGraw-HillBook Company, NY, 1986).

The fermentation can be any method of cultivation of a cell resulting inthe expression or isolation of an enzyme or protein. Fermentation may,therefore, be understood as comprising shake flask cultivation, orsmall- or large-scale fermentation (including continuous, batch,fed-batch, or solid state fermentations) in laboratory or industrialfermentors performed in a suitable medium and under conditions allowingthe enzyme to be expressed or isolated. The resulting enzymes producedby the methods described above may be recovered from the fermentationmedium and purified by conventional procedures.

Fermentation.

The fermentable sugars obtained from the hydrolyzed cellulosic materialcan be fermented by one or more (e.g., several) fermentingmicroorganisms capable of fermenting the sugars directly or indirectlyinto a desired fermentation product.

“Fermentation” or “fermentation process” refers to any fermentationprocess or any process comprising a fermentation step. Fermentationprocesses also include fermentation processes used in the consumablealcohol industry (e.g., beer and wine), dairy industry (e.g., fermenteddairy products), leather industry, and tobacco industry. Thefermentation conditions depend on the desired fermentation product andfermenting organism and can easily be determined by one skilled in theart.

In the fermentation step, sugars, released from the cellulosic materialas a result of the pretreatment and enzymatic hydrolysis steps, arefermented to a product, e.g., ethanol, by a fermenting organism, such asyeast. Hydrolysis (saccharification) and fermentation can be separate orsimultaneous, as described herein.

Any suitable hydrolyzed cellulosic material can be used in thefermentation step in practicing the present invention. The material isgenerally selected based on the desired fermentation product, i.e., thesubstance to be obtained from the fermentation, and the processemployed, as is well known in the art.

The term “fermentation medium” is understood herein to refer to a mediumbefore the fermenting microorganism(s) is(are) added, such as, a mediumresulting from a saccharification process, as well as a medium used in asimultaneous saccharification and fermentation process (SSF).

“Fermenting microorganism” refers to any microorganism, includingbacterial and fungal organisms, suitable for use in a desiredfermentation process to produce a fermentation product. The fermentingorganism can be hexose and/or pentose fermenting organisms, or acombination thereof. Both hexose and pentose fermenting organisms arewell known in the art. Suitable fermenting microorganisms are able toferment, i.e., convert, sugars, such as glucose, xylose, xylulose,arabinose, maltose, mannose, galactose, and/or oligosaccharides,directly or indirectly into the desired fermentation product. Examplesof bacterial and fungal fermenting organisms producing ethanol aredescribed by Lin et al., 2006, Appl. Microbiol. Biotechnol. 69: 627-642.

Examples of fermenting microorganisms that can ferment hexose sugarsinclude bacterial and fungal organisms, such as yeast. Preferred yeastincludes strains of Candida, Kluyveromyces, and Saccharomyces, e.g.,Candida sonorensis, Kluyveromyces marxianus, and Saccharomycescerevisiae.

Examples of fermenting organisms that can ferment pentose sugars intheir native state include bacterial and fungal organisms, such as someyeast. Preferred xylose fermenting yeast include strains of Candida,preferably C. sheatae or C. sonorensis; and strains of Pichia,preferably P. stipitis, such as P. stipitis CBS 5773. Preferred pentosefermenting yeast include strains of Pachysolen, preferably P.tannophilus. Organisms not capable of fermenting pentose sugars, such asxylose and arabinose, may be genetically modified to do so by methodsknown in the art.

Examples of bacteria that can efficiently ferment hexose and pentose toethanol include, for example, Bacillus coagulans, Clostridiumacetobutylicum, Clostridium thermocellum, Clostridium phytofermentans,Geobacillus sp., Thermoanaerobacter saccharolyticum, and Zymomonasmobilis (Philippidis, 1996, supra).

Other fermenting organisms include strains of Bacillus, such as Bacilluscoagulans; Candida, such as C. sonorensis, C. methanosorbosa, C.diddensiae, C. parapsilosis, C. naedodendra, C. blankii, C.entomophilia, C. brassicae, C. pseudotropicalis, C. boidinii, C. utilis,and C. scehatae; Clostridium, such as C. acetobutylicum, C.thermocellum, and C. phytofermentans; E. coli, especially E. colistrains that have been genetically modified to improve the yield ofethanol; Geobacillus sp.; Hansenula, such as Hansenula anomala;Klebsiella, such as K. oxytoca; Kluyveromyces, such as K. marxianus, K.lactis, K. thermotolerans, and K. fragilis; Schizosaccharomyces, such asS. pombe; Thermoanaerobacter, such as Thermoanaerobactersaccharolyticum; and Zymomonas, such as Zymomonas mobilis.

In a preferred aspect, the yeast is a Bretannomyces. In a more preferredaspect, the yeast is Bretannomyces clausenii. In another preferredaspect, the yeast is a Candida. In another more preferred aspect, theyeast is Candida sonorensis. In another more preferred aspect, the yeastis Candida boidinii. In another more preferred aspect, the yeast isCandida blankii. In another more preferred aspect, the yeast is Candidabrassicae. In another more preferred aspect, the yeast is Candidadiddensii. In another more preferred aspect, the yeast is Candidaentomophiliia. In another more preferred aspect, the yeast is Candidapseudotropicalis. In another more preferred aspect, the yeast is Candidascehatae. In another more preferred aspect, the yeast is Candida utilis.In another preferred aspect, the yeast is a Clavispora. In another morepreferred aspect, the yeast is Clavispora lusitaniae. In another morepreferred aspect, the yeast is Clavispora opuntiae. In another preferredaspect, the yeast is a Kluyveromyces. In another more preferred aspect,the yeast is Kluyveromyces fragilis. In another more preferred aspect,the yeast is Kluyveromyces marxianus. In another more preferred aspect,the yeast is Kluyveromyces thermotolerans. In another preferred aspect,the yeast is a Pachysolen. In another more preferred aspect, the yeastis Pachysolen tannophilus. In another preferred aspect, the yeast is aPichia. In another more preferred aspect, the yeast is a Pichiastipitis. In another preferred aspect, the yeast is a Saccharomyces spp.In a more preferred aspect, the yeast is Saccharomyces cerevisiae. Inanother more preferred aspect, the yeast is Saccharomyces distaticus. Inanother more preferred aspect, the yeast is Saccharomyces uvarum.

In a preferred aspect, the bacterium is a Bacillus. In a more preferredaspect, the bacterium is Bacillus coagulans. In another preferredaspect, the bacterium is a Clostridium. In another more preferredaspect, the bacterium is Clostridium acetobutylicum. In another morepreferred aspect, the bacterium is Clostridium phytofermentans. Inanother more preferred aspect, the bacterium is Clostridiumthermocellum. In another more preferred aspect, the bacterium isGeobacilus sp. In another more preferred aspect, the bacterium is aThermoanaerobacter. In another more preferred aspect, the bacterium isThermoanaerobacter saccharolyticum. In another preferred aspect, thebacterium is a Zymomonas. In another more preferred aspect, thebacterium is Zymomonas mobilis.

Commercially available yeast suitable for ethanol production include,e.g., BIOFERM™ AFT and XR (NABC—North American Bioproducts Corporation,GA, USA), ETHANOL RED™ yeast (Fermentis/Lesaffre, USA), FALI™(Fleischmann's Yeast, USA), FERMIOL™ (DSM Specialties), GERT STRAND™(Gert Strand AB, Sweden), and SUPERSTART™ and THERMOSACC™ fresh yeast(Ethanol Technology, WI, USA).

In a preferred aspect, the fermenting microorganism has been geneticallymodified to provide the ability to ferment pentose sugars, such asxylose utilizing, arabinose utilizing, and xylose and arabinoseco-utilizing microorganisms.

The cloning of heterologous genes into various fermenting microorganismshas led to the construction of organisms capable of converting hexosesand pentoses to ethanol (co-fermentation) (Chen and Ho, 1993, Cloningand improving the expression of Pichia stipitis xylose reductase gene inSaccharomyces cerevisiae, Appl. Biochem. Biotechnol. 39-40: 135-147; Hoet al., 1998, Genetically engineered Saccharomyces yeast capable ofeffectively cofermenting glucose and xylose, Appl. Environ. Microbiol.64: 1852-1859; Kotter and Ciriacy, 1993, Xylose fermentation bySaccharomyces cerevisiae, Appl. Microbiol. Biotechnol. 38: 776-783;Walfridsson et al., 1995, Xylose-metabolizing Saccharomyces cerevisiaestrains overexpressing the TKL1 and TAL1 genes encoding the pentosephosphate pathway enzymes transketolase and transaldolase, Appl.Environ. Microbiol. 61: 4184-4190; Kuyper et al., 2004, Minimalmetabolic engineering of Saccharomyces cerevisiae for efficientanaerobic xylose fermentation: a proof of principle, FEMS Yeast Research4: 655-664; Beall et al., 1991, Parametric studies of ethanol productionfrom xylose and other sugars by recombinant Escherichia coli, Biotech.Bioeng. 38: 296-303; Ingram et al., 1998, Metabolic engineering ofbacteria for ethanol production, Biotechnol. Bioeng. 58: 204-214; Zhanget al., 1995, Metabolic engineering of a pentose metabolism pathway inethanologenic Zymomonas mobilis, Science 267: 240-243; Deanda et al.,1996, Development of an arabinose-fermenting Zymomonas mobilis strain bymetabolic pathway engineering, Appl. Environ. Microbiol. 62: 4465-4470;WO 2003/062430, xylose isomerase).

In a preferred aspect, the genetically modified fermenting microorganismis Candida sonorensis. In another preferred aspect, the geneticallymodified fermenting microorganism is Escherichia coli. In anotherpreferred aspect, the genetically modified fermenting microorganism isKlebsiella oxytoca. In another preferred aspect, the geneticallymodified fermenting microorganism is Kluyveromyces marxianus. In anotherpreferred aspect, the genetically modified fermenting microorganism isSaccharomyces cerevisiae. In another preferred aspect, the geneticallymodified fermenting microorganism is Zymomonas mobilis.

It is well known in the art that the organisms described above can alsobe used to produce other substances, as described herein.

The fermenting microorganism is typically added to the degradedcellulosic material or hydrolysate and the fermentation is performed forabout 8 to about 96 hours, e.g., about 24 to about 60 hours. Thetemperature is typically between about 26° C. to about 60° C., e.g.,about 32° C. or 50° C., and about pH 3 to about pH 8, e.g., pH 4-5, 6,or 7.

In one aspect, the yeast and/or another microorganism are applied to thedegraded cellulosic material and the fermentation is performed for about12 to about 96 hours, such as typically 24-60 hours. In another aspect,the temperature is preferably between about 20° C. to about 60° C.,e.g., about 25° C. to about 50° C., about 32° C. to about 50° C., orabout 32° C. to about 50° C., and the pH is generally from about pH 3 toabout pH 7, e.g., about pH 4 to about pH 7. However, some fermentingorganisms, e.g., bacteria, have higher fermentation temperature optima.Yeast or another microorganism is preferably applied in amounts ofapproximately 10⁵ to 10¹², preferably from approximately 10⁷ to 10¹⁰,especially approximately 2×10⁸ viable cell count per ml of fermentationbroth. Further guidance in respect of using yeast for fermentation canbe found in, e.g., “The Alcohol Textbook” (Editors K. Jacques, T. P.Lyons and D. R. Kelsall, Nottingham University Press, United Kingdom1999), which is hereby incorporated by reference.

For ethanol production, following the fermentation the fermented slurryis distilled to extract the ethanol. The ethanol obtained according tothe processes of the invention can be used as, e.g., fuel ethanol,drinking ethanol, i.e., potable neutral spirits, or industrial ethanol.

A fermentation stimulator can be used in combination with any of theprocesses described herein to further improve the fermentation process,and in particular, the performance of the fermenting microorganism, suchas, rate enhancement and ethanol yield. A “fermentation stimulator”refers to stimulators for growth of the fermenting microorganisms, inparticular, yeast. Preferred fermentation stimulators for growth includevitamins and minerals. Examples of vitamins include multivitamins,biotin, pantothenate, nicotinic acid, meso-inositol, thiamine,pyridoxine, para-aminobenzoic acid, folic acid, riboflavin, and VitaminsA, B, C, D, and E. See, for example, Alfenore et al., Improving ethanolproduction and viability of Saccharomyces cerevisiae by a vitaminfeeding strategy during fed-batch process, Springer-Verlag (2002), whichis hereby incorporated by reference. Examples of minerals includeminerals and mineral salts that can supply nutrients comprising P, K,Mg, S, Ca, Fe, Zn, Mn, and Cu.

Fermentation Products:

A fermentation product can be any substance derived from thefermentation. The fermentation product can be, without limitation, analcohol (e.g., arabinitol, n-butanol, isobutanol, ethanol, glycerol,methanol, ethylene glycol, 1,3-propanediol [propylene glycol],butanediol, glycerin, sorbitol, and xylitol); an alkane (e.g., pentane,hexane, heptane, octane, nonane, decane, undecane, and dodecane), acycloalkane (e.g., cyclopentane, cyclohexane, cycloheptane, andcyclooctane), an alkene (e.g. pentene, hexene, heptene, and octene); anamino acid (e.g., aspartic acid, glutamic acid, glycine, lysine, serine,and threonine); a gas (e.g., methane, hydrogen (H₂), carbon dioxide(CO₂), and carbon monoxide (CO)); isoprene; a ketone (e.g., acetone); anorganic acid (e.g., acetic acid, acetonic acid, adipic acid, ascorbicacid, citric acid, 2,5-diketo-D-gluconic acid, formic acid, fumaricacid, glucaric acid, gluconic acid, glucuronic acid, glutaric acid,3-hydroxypropionic acid, itaconic acid, lactic acid, malic acid, malonicacid, oxalic acid, oxaloacetic acid, propionic acid, succinic acid, andxylonic acid); and polyketide. The fermentation product can also beprotein as a high value product.

In a preferred aspect, the fermentation product is an alcohol. It willbe understood that the term “alcohol” encompasses a substance thatcontains one or more hydroxyl moieties. In a more preferred aspect, thealcohol is n-butanol. In another more preferred aspect, the alcohol isisobutanol. In another more preferred aspect, the alcohol is ethanol. Inanother more preferred aspect, the alcohol is methanol. In another morepreferred aspect, the alcohol is arabinitol. In another more preferredaspect, the alcohol is butanediol. In another more preferred aspect, thealcohol is ethylene glycol. In another more preferred aspect, thealcohol is glycerin. In another more preferred aspect, the alcohol isglycerol. In another more preferred aspect, the alcohol is1,3-propanediol. In another more preferred aspect, the alcohol issorbitol. In another more preferred aspect, the alcohol is xylitol. See,for example, Gong, C. S., Cao, N. J., Du, J., and Tsao, G. T., 1999,Ethanol production from renewable resources, in Advances in BiochemicalEngineering/Biotechnology, Scheper, T., ed., Springer-Verlag BerlinHeidelberg, Germany, 65: 207-241; Silveira, M. M., and Jonas, R., 2002,The biotechnological production of sorbitol, Appl. Microbiol.Biotechnol. 59: 400-408; Nigam, P., and Singh, D., 1995, Processes forfermentative production of xylitol—a sugar substitute, ProcessBiochemistry 30 (2): 117-124; Ezeji, T. C., Qureshi, N. and Blaschek, H.P., 2003, Production of acetone, butanol and ethanol by Clostridiumbeijerinckii BA101 and in situ recovery by gas stripping, World Journalof Microbiology and Biotechnology 19 (6): 595-603.

In another preferred aspect, the fermentation product is an alkane. Thealkane can be an unbranched or a branched alkane. In another morepreferred aspect, the alkane is pentane. In another more preferredaspect, the alkane is hexane. In another more preferred aspect, thealkane is heptane. In another more preferred aspect, the alkane isoctane. In another more preferred aspect, the alkane is nonane. Inanother more preferred aspect, the alkane is decane. In another morepreferred aspect, the alkane is undecane. In another more preferredaspect, the alkane is dodecane.

In another preferred aspect, the fermentation product is a cycloalkane.In another more preferred aspect, the cycloalkane is cyclopentane. Inanother more preferred aspect, the cycloalkane is cyclohexane. Inanother more preferred aspect, the cycloalkane is cycloheptane. Inanother more preferred aspect, the cycloalkane is cyclooctane.

In another preferred aspect, the fermentation product is an alkene. Thealkene can be an unbranched or a branched alkene. In another morepreferred aspect, the alkene is pentene. In another more preferredaspect, the alkene is hexene. In another more preferred aspect, thealkene is heptene. In another more preferred aspect, the alkene isoctene.

In another preferred aspect, the fermentation product is an amino acid.In another more preferred aspect, the organic acid is aspartic acid. Inanother more preferred aspect, the amino acid is glutamic acid. Inanother more preferred aspect, the amino acid is glycine. In anothermore preferred aspect, the amino acid is lysine. In another morepreferred aspect, the amino acid is serine. In another more preferredaspect, the amino acid is threonine. See, for example, Richard, A., andMargaritis, A., 2004, Empirical modeling of batch fermentation kineticsfor poly(glutamic acid) production and other microbial biopolymers,Biotechnology and Bioengineering 87 (4): 501-515.

In another preferred aspect, the fermentation product is a gas. Inanother more preferred aspect, the gas is methane. In another morepreferred aspect, the gas is H₂. In another more preferred aspect, thegas is CO₂. In another more preferred aspect, the gas is CO. See, forexample, Kataoka, N., A. Miya, and K. Kiriyama, 1997, Studies onhydrogen production by continuous culture system of hydrogen-producinganaerobic bacteria, Water Science and Technology 36 (6-7): 41-47; andGunaseelan V. N. in Biomass and Bioenergy, Vol. 13 (1-2), pp. 83-114,1997, Anaerobic digestion of biomass for methane production: A review.

In another preferred aspect, the fermentation product is isoprene.

In another preferred aspect, the fermentation product is a ketone. Itwill be understood that the term “ketone” encompasses a substance thatcontains one or more ketone moieties. In another more preferred aspect,the ketone is acetone. See, for example, Qureshi and Blaschek, 2003,supra.

In another preferred aspect, the fermentation product is an organicacid. In another more preferred aspect, the organic acid is acetic acid.In another more preferred aspect, the organic acid is acetonic acid. Inanother more preferred aspect, the organic acid is adipic acid. Inanother more preferred aspect, the organic acid is ascorbic acid. Inanother more preferred aspect, the organic acid is citric acid. Inanother more preferred aspect, the organic acid is 2,5-diketo-D-gluconicacid. In another more preferred aspect, the organic acid is formic acid.In another more preferred aspect, the organic acid is fumaric acid. Inanother more preferred aspect, the organic acid is glucaric acid. Inanother more preferred aspect, the organic acid is gluconic acid. Inanother more preferred aspect, the organic acid is glucuronic acid. Inanother more preferred aspect, the organic acid is glutaric acid. Inanother preferred aspect, the organic acid is 3-hydroxypropionic acid.In another more preferred aspect, the organic acid is itaconic acid. Inanother more preferred aspect, the organic acid is lactic acid. Inanother more preferred aspect, the organic acid is malic acid. Inanother more preferred aspect, the organic acid is malonic acid. Inanother more preferred aspect, the organic acid is oxalic acid. Inanother more preferred aspect, the organic acid is propionic acid. Inanother more preferred aspect, the organic acid is succinic acid. Inanother more preferred aspect, the organic acid is xylonic acid. See,for example, Chen, R., and Lee, Y. Y., 1997, Membrane-mediatedextractive fermentation for lactic acid production from cellulosicbiomass, Appl. Biochem. Biotechnol. 63-65: 435-448.

In another preferred aspect, the fermentation product is polyketide.

Recovery.

The fermentation product(s) can be optionally recovered from thefermentation medium using any method known in the art including, but notlimited to, chromatography, electrophoretic procedures, differentialsolubility, distillation, or extraction. For example, alcohol isseparated from the fermented cellulosic material and purified byconventional methods of distillation. Ethanol with a purity of up toabout 96 vol. % can be obtained, which can be used as, for example, fuelethanol, drinking ethanol, i.e., potable neutral spirits, or industrialethanol.

Signal Peptides

The present invention also relates to an isolated polynucleotideencoding a signal peptide comprising or consisting of amino acids 1 to20 of SEQ ID NO: 2, amino acids 1 to 25 of SEQ ID NO: 4, amino acids 1to 20 of SEQ ID NO: 6, amino acids 1 to 21 of SEQ ID NO: 8, amino acids1 to 19 of SEQ ID NO: 10, amino acids 1 to 17 of SEQ ID NO: 12, or aminoacids 1 to 20 of SEQ ID NO: 14. The polynucleotides may further comprisea gene encoding a protein, which is operably linked to the signalpeptide. The protein is preferably foreign to the signal peptide.

The present invention also relates to nucleic acid constructs,expression vectors and recombinant host cells comprising suchpolynucleotides.

The present invention also relates to methods of producing a protein,comprising: (a) cultivating a recombinant host cell comprising suchpolynucleotide; and (b) recovering the protein.

The protein may be native or heterologous to a host cell. The term“protein” is not meant herein to refer to a specific length of theencoded product and, therefore, encompasses peptides, oligopeptides, andpolypeptides. The term “protein” also encompasses two or morepolypeptides combined to form the encoded product. The proteins alsoinclude hybrid polypeptides and fused polypeptides.

Preferably, the protein is a hormone or variant thereof, enzyme,receptor or portion thereof, antibody or portion thereof, or reporter.For example, the protein may be an oxidoreductase, transferase,hydrolase, lyase, isomerase, or ligase such as an aminopeptidase,amylase, carbohydrase, carboxypeptidase, catalase, cellulase, chitinase,cutinase, cyclodextrin glycosyltransferase, deoxyribonuclease, esterase,alpha-galactosidase, beta-galactosidase, glucoamylase,alpha-glucosidase, beta-glucosidase, invertase, laccase, another lipase,mannosidase, mutanase, oxidase, pectinolytic enzyme, peroxidase,phytase, polyphenoloxidase, proteolytic enzyme, ribonuclease,transglutaminase or xylanase.

The gene may be obtained from any prokaryotic, eukaryotic, or othersource.

The present invention is further described by the following examplesthat should not be construed as limiting the scope of the invention.

EXAMPLES

Materials

Chemicals used as buffers and substrates were commercial products of atleast reagent grade.

Strains

Aspergillus aculeatus CBS 172.66 was used as the source of polypeptideshaving cellulolytic enhancing activity. Aspergillus oryzae MT3568 strainwas used for expression of the A. aculeatus genes encoding thepolypeptides having cellulolytic enhancing activity. A. oryzae MT3568 isan amdS (acetamidase) disrupted gene derivative of Aspergillus oryzaeJaL355 (WO 2002/40694) in which pyrG auxotrophy was restored bydisrupting the A. oryzae acetamidase (amdS) gene with the pyrG gene.

Media and Solutions

YP+2% glucose medium was composed of 1% yeast extract, 2% peptone and 2%glucose.

YP+2% maltodextrin medium was composed of 1% yeast extract, 2% peptoneand 2% maltodextrin.

PDA agar plates were composed of potato infusion (potato infusion wasmade by boiling 300 g of sliced (washed but unpeeled) potatoes in waterfor 30 minutes and then decanting or straining the broth throughcheesecloth. Distilled water was then added until the total volume ofthe suspension was one liter, followed by 20 g of dextrose and 20 g ofagar powder. The medium was sterilized by autoclaving at 15 psi for 15minutes (Bacteriological Analytical Manual, 8th Edition, Revision A,1998).

LB plates were composed of 10 g of Bacto-Tryptone, 5 g of yeast extract,10 g of sodium chloride, 15 g of Bacto-agar, and deionized water to 1liter.

LB medium was composed of 10 g of Bacto-Tryptone, 5 g of yeast extract,10 g of sodium chloride, and deionized water to 1 liter.

COVE sucrose plates were composed of 342 g of sucrose, 20 g of agarpowder, 20 ml of COVE salts solution, and deionized water to 1 liter.The medium was sterilized by autoclaving at 15 psi for 15 minutes(Bacteriological Analytical Manual, 8th Edition, Revision A, 1998). Themedium was cooled to 60° C. and 10 mM acetamide, 15 mM CsCl, TRITON®X-100 (50 μl/500 ml) were added.

COVE salts solution was composed of 26 g of MgSO₄.7H₂O, 26 g of KCL, 26g of KH₂PO₄, 50 ml of COVE trace metals solution, and deionized water to1 liter.

COVE trace metals solution was composed of 0.04 g of Na₂B₄O₇.10H₂O, 0.4g of CuSO₄.5H₂O, 1.2 g of FeSO₄.7H₂O, 0.7 g of MnSO₄.H₂O, 0.8 g ofNa₂MoO₄.2H₂O, 10 g of ZnSO₄.7H₂O, and deionized water to 1 liter.

Example 1 Source of DNA Sequence Information for Aspergillus aculeatusCBS 172.66

Genomic sequence information was generated by the U.S. Department ofEnergy Joint Genome Institute (JGI). A preliminary assembly of thegenome was downloaded from JGI and analyzed using the Pedant-Pro™Sequence Analysis Suite (Biomax Informatics AG, Martinsried, Germany).Gene models constructed by the software were used as a starting pointfor detecting GH61 homologues in the genome. More precise gene modelswere constructed manually using multiple known GH61 protein sequences asa guide.

Example 2 Aspergillus aculeatus CBS 172.66 Genomic DNA Extraction

Aspergillus aculeatus CBS 172.66 was propagated on PDA agar plates at26° C. for 7 days. Spores harvested from the PDA plates were used toinoculate 25 ml of YP+2% glucose medium in a baffled shake flask andincubated at 26° C. for 48 hours with agitation at 200 rpm.

Genomic DNA was isolated according to a modified FASTDNA® SPIN protocol(Qbiogene, Inc., Carlsbad, Calif., USA). Briefly a FASTDNA® SPIN Kit forSoil (Qbiogene, Inc., Carlsbad, Calif., USA) was used in a FASTPREP® 24Homogenization System (MP Biosciences, Santa Ana, Calif., USA). Two mlof fungal material from the above culture was harvested bycentrifugation at 14,000×g for 2 minutes. The supernatant was removedand the pellet resuspended in 500 μl of deionized water. The suspensionwas transferred to a Lysing Matrix E FASTPREP® tube (Qbiogene, Inc.,Carlsbad, Calif., USA) and 790 μl of sodium phosphate buffer and 100 μlof MT buffer from the FASTDNA® SPIN Kit were added to the tube. Thesample was then secured in the FASTPREP® Instrument (Qbiogene, Inc.,Carlsbad, Calif., USA) and processed for 60 seconds at a speed of 5.5m/sec. The sample was then centrifuged at 14,000×g for two minutes andthe supernatant transferred to a clean EPPENDORF® tube. A 250 μl volumeof PPS reagent from the FASTDNA® SPIN Kit was added and then the samplewas mixed gently by inversion. The sample was again centrifuged at14,000×g for 5 minutes. The supernatant was transferred to a 15 ml tubefollowed by 1 ml of Binding Matrix suspension from the FASTDNA® SPIN Kitand then mixed by inversion for two minutes. The sample was placed in astationary tube rack and the silica matrix was allowed to settle for 3minutes. A 500 μl volume of the supernatant was removed and discardedand then the remaining sample was resuspended in the matrix. The samplewas then transferred to a SPIN filter tube from the FASTDNA® SPIN Kitand centrifuged at 14,000×g for 1 minute. The catch tube was emptied andthe remaining matrix suspension added to the SPIN filter tube. Thesample was again centrifuged at 14,000×g for 1 minute. A 500 μl volumeof SEWS-M solution from the FASTDNA® SPIN Kit was added to the SPINfilter tube and the sample was centrifuged at the same speed for 1minute. The catch tube was emptied and the SPIN filter replaced in thecatch tube. The unit was centrifuged at 14,000×g for 2 minutes to drythe matrix of residual SEWS-M wash solution. The SPIN filter was placedin a fresh catch tube and allowed to air dry for 5 minutes at roomtemperature. The matrix was gently resuspended in 100 μl of DES(DNase/Pyrogen free water) with a pipette tip. The unit was centrifugedat 14,000×g for 1 minute to elute the genomic DNA followed by elutionwith 100 μl of 0.1 mM EDTA-10 mM Tris pH 8.0 by centrifugation at14,000×g for 1 minute and the eluates were combined. The concentrationof the DNA harvested from the catch tube was measured at 260 nm with aUV spectrophotometer.

Example 3 Construction of an Aspergillus oryzae Expression VectorContaining Aspergillus aculeatus CBS 172.66 Genomic Sequence Encoding aGH61 Polypeptide Having Cellulolytic Enhancing Activity

Two synthetic oligonucleotide primers shown below were designed toamplify by PCR the Aspergillus aculeatus CBS 172.66 P23NNT GH61 genefrom the genomic DNA prepared in Example 2. An IN-FUSION® Cloning Kit(Clontech Laboratories, Inc., Mountain View, Calif., USA) was used toclone the fragment directly into the expression vector pDau109 (WO2005/042735).

Primer F-P23NNT: (SEQ ID NO: 15)5′-ACACAACTGGGGATCCACCATGAAGTATATTCCTCTCGTTAT-3′ Primer R-P23NNT:(SEQ ID NO: 16) 5′-AGATCTCGAGAAGCTTAGCACGAATTCACGCATTGAT-3′Bold letters represent coding sequence. The underlined sequence ishomologous to insertion sites of pDau109.

The PCR reaction was composed of 20 μl of Extensor Long PCR Master Mix(AB Gene Ltd., Epsom United Kingdom), Buffer 1 (AB Gene Ltd., EpsomUnited Kingdom), REDDYMIX™ Version (AB Gene Ltd., Epsom United Kingdom),1 μl of primer F—P23NN (100 μM), 1 μl of primer R—P23NNT (100 μM), 1 μlof A. aculeatus genomic DNA (100 ng/μl of 1 mM EDTA-10 mM Tris pH 8.0),and 17 μl of deionized water in a total volume of 40 μl. The master mixcontains buffer, dNTPs and a thermostable DNA polymerase blend. The PCRwas performed in a PTC-200 DNA engine (MJ Research Inc., Waltham, Mass.,USA) programmed for 1 cycle at 95° C. for 2 minutes. 30 cycles each at94° C. for 10 seconds, 55° C. for 30 seconds, and 68° C. for 1 minute;and 1 cycle at 68° C. for 10 minutes. The sample was then held at 10° C.until removed from the PCR machine.

The reaction products were isolated by 1.0% agarose gel electrophoresisusing 40 mM Tris base, 20 mM sodium acetate, 1 mM disodium EDTA (TAE)buffer where a 1068 bp product band was excised from the gel andpurified using an illustra GFX® PCR DNA and Gel Band Purification Kit(GE Healthcare Life Sciences, Brondby, Denmark) according to themanufacturer's instructions. The fragment was then cloned into Hind IIIand Bam HI digested pDau109 using an IN-FUSION® Cloning Kit resulting inplasmid pP23NNT. Cloning of the P23NNT gene into Hind III-Bam HIdigested pDau109 resulted in transcription of the Aspergillus aculeatusP23NNT gene under the control of a NA2-tpi double promoter. The NA2-tpipromoter is a modified promoter from the gene encoding the Aspergillusniger neutral alpha-amylase in which the untranslated leader has beenreplaced by an untranslated leader from the gene encoding theAspergillus nidulans triose phosphate isomerase.

The cloning protocol was performed according to the IN-FUSION® CloningKit instructions generating a P23NNT GH61 construct. The treated plasmidand insert were transformed into FUSION BLUE™ E. coli cells (ClontechLaboratories, Inc., Mountain View, Calif., USA) according to themanufacturer's protocol and plated onto LB plates supplemented with 50μg of ampicillin per ml. After incubating at 37° C. overnight, colonieswere observed growing under selection on the LB ampicillin plates. Tencolonies transformed with the P23NNT GH61 construct were cultivated inLB medium supplemented with 50 μg of ampicillin per ml and plasmid DNAwas isolated using a JETQUICK™ Plasmid Purification Spin Kit (GENOMEDGmbH, Löhne, Germany) according to the manufacturer's instructions.

The isolated plasmids were sequenced with vector primers and P23NNT genespecific primers in order to determine a representative plasmidexpression clone that was free of PCR errors.

The pairs of synthetic oligonucleotide primers shown below were designedto PCR amplify the A. aculeatus CBS 172.66 P23NK1 GH61 gene, P23NJS GH61gene, P23NNN GH61 gene, and P23NHW GH61 gene from the A. aculeatusgenomic DNA using the same procedure described above for the P23NNT GH61gene.

P23NK1 GH61 gene Primer F-P23NK1: (SEQ ID NO: 19)5′-ACACAACTGGGGATCCACCATGAAGTCCTCTACTTTCGGT-3′ Primer R-P23NK1:(SEQ ID NO: 20) 5′-AGATCTCGAGAAGCTT AGGCAGACTCAACGCACTG-3′P23NJS GH61 gene Primer F-P23NJS: (SEQ ID NO: 21)5′-ACACAACTGGGGATCCACCATGCGTCAGGCTCAGTCTTT-3′ Primer R-P23NJS:(SEQ ID NO: 22) 5′-AGATCTCGAGAAGCTT AGGCAGAGATGCACTGGTAG-3′P23NNN GH61 gene Primer F-P23NNN: (SEQ ID NO: 23)5′-ACACAACTGGGGATCCACCATGTCTCTTTCCAAGATTGCC-3′ Primer R-P23NNN:(SEQ ID NO: 24) 5′-AGATCTCGAGAAGCTT AGTGACGCTTGGGACGCG-3′P23NHW GH61 gene Primer F-P23NHW: (SEQ ID NO: 25)5′-ACACAACTGGGGATCCACCATGCTTGTCAAACTCATCTCTT-3′ Primer R-P23NHW:(SEQ ID NO: 26) 5′-AGATCTCGAGAAGCTT AAGCCGTGGTTGTCACTGC-3′Bold letters represent coding sequence. The underlined sequence ishomologous to insertion sites of pDau109.

The pairs of synthetic oligonucleotide primers shown below were designedto PCR amplify the A. aculeatus CBS 172.66 P23NJ4 gene and P23NQ3 GH61gene from the A. aculeatus genomic DNA using the same proceduredescribed above for the P23NNT GH61 gene.

P23NJ4 GH61 gene Primer F-P23NJ4: (SEQ ID NO: 17)5′-ACACAACTGGGGATCCACCATGTCTGTTGCTAAGTTTGCT-3′ Primer R-P23NJ4:(SEQ ID NO: 18) 5′-AGATCTCGAGAAGCTT AGGAGAGGTCACGGGCGT-3′P23NQ3 GH61 gene Primer F-P23NQ3: (SEQ ID NO: 27)5′-ACACAACTGGGGATCCACCATGAAGTATCTTGCGATCTTC-3′ Primer R-P23NQ3:(SEQ ID NO: 28) AGATCTCGAGAAGCTT ATCAATAAACACGACAAGCATG-3′Bold letters represent coding sequence. The underlined sequence ishomologous to the insertion sites of pDau109.

Example 4 Characterization of Aspergillus aculeatus CBS 172.66 GenomicSequences Encoding GH61 Polypeptides Having Cellulolytic-EnhancingActivity

DNA sequencing of the Aspergillus aculeatus CBS 172.66 GH61 genomicclones was performed with an Applied Biosystems Model 3700 Automated DNASequencer using version 3.1 BIG-DYE™ terminator chemistry (AppliedBiosystems, Inc., Foster City, Calif., USA) and primer walking strategy.Nucleotide sequence data were scrutinized for quality and all sequenceswere compared to each other with assistance of PHRED/PHRAP software(University of Washington, Seattle, Wash., USA).

The nucleotide sequence and deduced amino acid sequence of theAspergillus aculeatus P23NNT GH61 gene are shown in SEQ ID NO: 1 and SEQID NO: 2, respectively. The coding sequence is 1200 bp including thestop codon and is interrupted by introns of 85 bp (nucleotides 332 to416) and 80 bp (nucleotides 661 to 740). The encoded predicted proteinis 344 amino acids. Using the SignalP program (Nielsen et al., 1997,Protein Engineering 10:1-6), a signal peptide of 20 residues waspredicted. The predicted mature protein contains 324 amino acids.

A comparative pairwise global alignment of amino acid sequences wasdetermined using the Needleman and Wunsch algorithm (Needleman andWunsch, 1970, J. Mol. Biol. 48: 443-453) with gap open penalty of 10,gap extension penalty of 0.5, and the EBLOSUM62 matrix. The alignmentshowed that the deduced amino acid sequence of the Aspergillus aculeatusgene encoding the P23NNT GH61 polypeptide shares 18% identity (excludinggaps) to the deduced amino acid sequence of a GH61 family protein fromThermoascus aurantiacus (accession number GENESEQP:AEC05922).

The nucleotide sequence and deduced amino acid sequence of theAspergillus aculeatus P23NJ4 GH61 gene are shown in SEQ ID NO: 3 and SEQID NO: 4, respectively. The coding sequence is 1203 bp including thestop codon and contains no introns. The encoded predicted protein is 400amino acids. Using the SignalP program (Nielsen et al., 1997, supra), asignal peptide of 25 residues was predicted. The predicted matureprotein contains 375 amino acids.

A comparative pairwise global alignment of amino acid sequences wasdetermined using the Needleman and Wunsch algorithm (Needleman andWunsch, 1970, supra) with gap open penalty of 10, gap extension penaltyof 0.5, and the EBLOSUM62 matrix. The alignment showed that the deducedamino acid sequence of the Aspergillus aculeatus gene encoding theP23NJ4 GH61 polypeptide shares 73.8% identity (excluding gaps) to thededuced amino acid sequence of a GH61 family protein from Thermoascusaurantiacus (accession number GENESEQP:AEC05922).

The nucleotide sequence and deduced amino acid sequence of theAspergillus aculeatus P23NK1 GH61 gene are shown in SEQ ID NO: 5 and SEQID NO: 6, respectively. The coding sequence is 1170 bp including thestop codon and contains no introns. The encoded predicted protein is 389amino acids. Using the SignalP program (Nielsen et al., 1997, supra), asignal peptide of 20 residues was predicted. The predicted matureprotein contains 369 amino acids.

A comparative pairwise global alignment of amino acid sequences wasdetermined using the Needleman and Wunsch algorithm (Needleman andWunsch, 1970, supra) with gap open penalty of 10, gap extension penaltyof 0.5, and the EBLOSUM62 matrix. The alignment showed that the deducedamino acid sequence of the Aspergillus aculeatus gene encoding theP23NK1 GH61 polypeptide shares 32% identity (excluding gaps) to thededuced amino acid sequence of a GH61 family protein from Thermoascusaurantiacus (accession number GENESEQP:AEC05922).

The nucleotide sequence and deduced amino acid sequence of theAspergillus aculeatus P23NJS GH61 gene are shown in SEQ ID NO: 7 and SEQID NO: 8, respectively. The coding sequence is 1221 bp including thestop codon and contains no introns. The encoded predicted protein is 406amino acids. Using the SignalP program (Nielsen et al., 1997, supra), asignal peptide of 21 residues was predicted. The predicted matureprotein contains 385 amino acids.

A comparative pairwise global alignment of amino acid sequences wasdetermined using the Needleman and Wunsch algorithm (Needleman andWunsch, 1970, supra) with gap open penalty of 10, gap extension penaltyof 0.5, and the EBLOSUM62 matrix. The alignment showed that the deducedamino acid sequence of the Aspergillus aculeatus gene encoding theP23NJS GH61 polypeptide shares 60% identity (excluding gaps) to thededuced amino acid sequence of a GH61 family protein from Thermoascusaurantiacus (accession number GENESEQP:AEC05922).

The nucleotide sequence and deduced amino acid sequence of theAspergillus aculeatus P23NNN GH61 gene are shown in SEQ ID NO: 9 and SEQID NO: 10, respectively. The coding sequence is 1284 bp including thestop codon and contains no introns. The encoded predicted protein is 427amino acids. Using the SignalP program (Nielsen et al., 1997, supra), asignal peptide of 19 residues was predicted. The predicted matureprotein contains 408 amino acids.

A comparative pairwise global alignment of amino acid sequences wasdetermined using the Needleman and Wunsch algorithm (Needleman andWunsch, 1970, supra) with gap open penalty of 10, gap extension penaltyof 0.5, and the EBLOSUM62 matrix. The alignment showed that the deducedamino acid sequence of the Aspergillus aculeatus gene encoding theP23NNN GH61 polypeptide shares 62% identity (excluding gaps) to thededuced amino acid sequence of a GH61 family protein from Thermoascusaurantiacus (accession number GENESEQP:AEC05922).

The nucleotide sequence and deduced amino acid sequence of theAspergillus aculeatus P23NHW GH61 gene are shown in SEQ ID NO: 11 andSEQ ID NO: 12, respectively. The coding sequence is 804 bp including thestop codon and contains no introns. The encoded predicted protein is 267amino acids. Using the SignalP program (Nielsen et al., 1997, supra), asignal peptide of 17 residues was predicted. The predicted matureprotein contains 250 amino acids.

A comparative pairwise global alignment of amino acid sequences wasdetermined using the Needleman and Wunsch algorithm (Needleman andWunsch, 1970, supra) with gap open penalty of 10, gap extension penaltyof 0.5, and the EBLOSUM62 matrix. The alignment showed that the deducedamino acid sequence of the Aspergillus aculeatus gene encoding theP23NHW GH61 polypeptide shares 59% identity (excluding gaps) to thededuced amino acid sequence of a GH61 family protein from Thermoascusaurantiacus (accession number GENESEQP:AEC05922).

The nucleotide sequence and deduced amino acid sequence of theAspergillus aculeatus P23NQ3 GH61 gene are shown in SEQ ID NO: 13 andSEQ ID NO: 14, respectively. The coding sequence is 822 bp including thestop codon and is interrupted by introns of 95 bp (nucleotides 332 to426) and 64 bp (nucleotides 671 to 734). The encoded predicted proteinis 273 amino acids. Using the SignalP program (Nielsen et al., 1997,supra), a signal peptide of 20 residues was predicted. The predictedmature protein contains 253 amino acids.

A comparative pairwise global alignment of amino acid sequences wasdetermined using the Needleman and Wunsch algorithm (Needleman andWunsch, 1970, supra) with gap open penalty of 10, gap extension penaltyof 0.5, and the EBLOSUM62 matrix. The alignment showed that the deducedamino acid sequence of the Aspergillus aculeatus gene encoding theP23NQ3 GH61 polypeptide shares 18% identity (excluding gaps) to thededuced amino acid sequence of a GH61 family protein from Thermoascusaurantiacus (accession number GENESEQP:AEC05922).

Example 5 Expression of Aspergillus aculeatus CBS 172.66 Family 61Glycosyl Hydrolase P23NNT Gene in Aspergillus oryzae MT3568

One error-free pP23NNT clone comprising the P23NNT GH61 gene of SEQ IDNO: 1 was selected for further work. Plasmid DNA was then isolated usinga JETSTAR 2.0 Plasmid Mini/Midi/Maxi-Protocol (Genomed). The purifiedplasmid DNA was transformed into protoplasts of Aspergillus oryzaeMT3568, which were prepared according to the method of Christensen etal., 1988, Bio/Technology 6: 1419-1422. The selection plates consistedof COVE sucrose+10 mM acetamide+15 mM CsCl+TRITON® X-100 (50 μl/500 ml).The plates were incubated at 37° C. The transformation of A. oryzaeMT3568 with pP23NNT yielded several transformants. Eight transformantswere inoculated into separate wells of a 96 microtiter deep well plate(Nunc A/S, Roskilde, Denmark) with each well containing 750 μl of YP+2%glucose medium or 750 μl of YP+2% maltodextrin. The plate was coveredwith Nunc prescored vinyl sealing tape (ThermoFisher Scientific,Roskilde, Denmark) and incubated at 26° C. stationary for 4 days. Thecolonies on the selection plates were also restreaked onto COVE sucrose(+10 mM acetamide+15 mM CsCl+TRITON® X-100 (50 μl/500 ml)). The plateswere incubated at 37° C. and this selection procedure was repeated inorder to stabilize the transformants.

Several Aspergillus oryzae transformants produced the recombinant P23NNTGH61 polypeptide of SEQ ID NO: 2 as judged by SDS-PAGE analysis.

Error-free clones for pP23NJS comprising the P23NJS GH61 gene, pP23NNNcomprising the P23NNN GH61 gene, pP23NK1 comprising the P23NK1 GH61gene, pP23NHW comprising the P23NHW GH61 gene, pP23NJ4 comprising theP23NJ4 GH61 gene, and pP23NQ3 comprising the P23NQ3 GH61 gene weretransformed into A. oryzae MT3568 using the same procedure above.

One Aspergillus oryzae transformant for each of the P23NHW GH61 gene(EXP03690), P23NJS GH61 gene (EXP03691), P23NNN GH61 gene (EXP03692),and P23NK1 GH61 gene (EXP03693) was streaked onto PDA plates andincubated at 34° C. for two weeks. Spores were removed in 5 ml of 0.01%TWEEN® 20 and 4 ml of spore suspension was inoculated into 500 ml ofYP+2% glucose medium, and incubated at 28° C. for 4 days with shaking at250 rpm. The supernatants were harvested by filtering through a 0.22 μmfilter.

Example 6 Effect of Aspergillus aculeatus GH61 Polypeptides onHydrolysis of Pretreated Corn Stover

The filtered supernatants (Example 5) from the Aspergillus oryzaetransformants for the P23NHW GH61 gene (EXP03690), P23NJS GH61 gene(EXP03691), P23NNN GH61 gene (EXP03692), and P23NK1 GH61 gene (EXP03693)were concentrated approximately 20-fold using an Amicon ultrafiltrationdevice (Millipore, Bedford, Mass., USA, 10 kDa polyethersulfonemembrane, 40 psi, 4° C.). Protein concentration was determined by a BCAProtein Assay (BCA Protein Assay Kit, Thermo Fisher Scientific, Waltham,Mass., USA). Corn stover was pretreated and prepared as an assaysubstrate as described in WO 2005/074647 to generate pretreated cornstover (PCS). The base cellulase mixture used to assay enhancingactivity was prepared from Trichoderma reesei strain SMA135 (WO2008/057637).

Hydrolysis of PCS was conducted using 1.6 ml deep-well plates (Axygen,Santa Clara, Calif., USA) with a total reaction volume of 1.0 ml and aPCS concentration of 50 mg/ml in 1 mM manganese sulfate-50 mM sodiumacetate pH 5.0. The Aspergillus aculeatus GH61 polypeptide wasseparately added to the base cellulase mixture at concentrations rangingfrom 0 to 100% of the protein concentration of the base cellulasemixture. Incubation was at 50° C. for 72 hours. Assays were performed intriplicate. Aliquots were centrifuged, and the supernatant liquid wasfiltered by centrifugation (MULTISCREEN® HV 0.45 μm, Millipore,Billerica, Mass., USA) at 3000 rpm for 10 minutes using a platecentrifuge (SORVALL® RT7, Thermo Fisher Scientific, Waltham, Mass.,USA). When not used immediately, filtered hydrolysate aliquots werefrozen at −20° C. Sugar concentrations of samples diluted in 0.005 MH₂SO₄ with 0.05% w/w benzoic acid were measured after elution by 0.005 MH₂SO₄ with 0.05% w/w benzoic acid at a flow rate of 0.6 ml/minute from a4.6×250 mm AMINEX® HPX-87H column (Bio-Rad Laboratories, Inc., Hercules,Calif., USA) at 65° C. with quantitation by integration of glucose andcellobiose signals from refractive index detection (CHEMSTATION®,AGILENT® 1100 HPLC, Agilent Technologies, Santa Clara, Calif., USA)calibrated by pure sugar samples (Absolute Standards Inc., Hamden,Conn., USA). The resultant equivalents were used to calculate thepercentage of cellulose conversion for each reaction. The degree ofcellulose conversion to glucose plus cellobiose sugars (conversion, %)was calculated using the following equation:Conversion (%)=(glucose+cellobiose×1.053) (mg/ml)×100×162/(Cellulose(mg/ml)×180)=(glucose+cellobiose×1.053) (mg/ml)×100/(Cellulose(mg/ml)×1.111)

In this equation the factor 1.111 reflects the weight gain in convertingcellulose to glucose, and the factor 1.053 reflects the weight gain inconverting cellobiose to glucose. Cellulose in PCS was determined by alimit digest of PCS to release glucose and cellobiose.

The result of adding increasing amounts of the Aspergillus aculeatusP23NHW GH61 polypeptide (EXP03690) to the base cellulase mix are shownin FIG. 1. Addition of the Aspergillus aculeatus GH61 polypeptideprovided a stimulation factor of 1.33 at a 100% addition level.

The result of adding increasing amounts of the Aspergillus aculeatusP23NJS GH61 polypeptide (EXP03691) to the base cellulase mix are shownin FIG. 2. Addition of the Aspergillus aculeatus GH61 polypeptideprovided a stimulation factor of 1.28 at a 100% addition level.

The result of adding increasing amounts of the Aspergillus aculeatusP23NNN GH61 polypeptide (EXP03692) to the base cellulase mix are shownin FIG. 3. Addition of the Aspergillus aculeatus GH61 polypeptideprovided a stimulation factor of 1.09 at a 100% addition level.

The result of adding increasing amounts of the Aspergillus aculeatusP23NK1 GH61 polypeptide (EXP03693) to the base cellulase mix are shownin FIG. 4. Addition of the Aspergillus aculeatus GH61 polypeptideprovided a stimulation factor of 1.05 at a 100% addition level.

The present invention is further described by the following numberedparagraphs:

[1] An isolated polypeptide having cellulolytic enhancing activity,selected from the group consisting of: (a) a polypeptide having at least60% sequence identity to the mature polypeptide of SEQ ID NO: 2, SEQ IDNO: 6, SEQ ID NO: 12, or SEQ ID NO: 14; at least 65% sequence identityto the mature polypeptide of SEQ ID NO: 8 or SEQ ID NO: 10, or at least75% sequence identity to the mature polypeptide of SEQ ID NO: 4; (b) apolypeptide encoded by a polynucleotide that hybridizes under at leastmedium-high stringency conditions with (i) the mature polypeptide codingsequence of SEQ ID NO: 1 or the cDNA sequence thereof, SEQ ID NO: 3, SEQID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, or SEQ ID NO: 13 orthe cDNA sequence thereof, or (ii) the full-length complement of (i);(c) a polypeptide encoded by a polynucleotide having at least 60%sequence identity to the mature polypeptide coding sequence of SEQ IDNO: 1 or the cDNA sequence thereof, SEQ ID NO: 5, SEQ ID NO: 11, or SEQID NO: 13, or the cDNA sequence thereof; at least 65% sequence identityto the mature polypeptide coding sequence of SEQ ID NO: 7 or SEQ ID NO:9; or at least 75% sequence identity to the mature polypeptide codingsequence of SEQ ID NO: 3; (d) a variant of the mature polypeptide of SEQID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQID NO: 12, or SEQ ID NO: 14 comprising a substitution, deletion, and/orinsertion at one or more (e.g., several) positions; and (e) a fragmentof the polypeptide of (a), (b), (c), or (d) that has cellulolyticenhancing activity.

[2] The polypeptide of paragraph 1, having a polypeptide having at least60%, at least 65%, at least 70%, at least 75%, at least 80%, at least81%, at least 83%, at least 83%, at least 84%, at least 85%, at least86%, at least 87%, at least 88%, at least 89%, at least 90%, at least91%, at least 92%, at least 93%, at least 94%, at least 95%, at least96%, at least 97%, at least 98%, at least 99%, or 100% sequence identityto the mature polypeptide of SEQ ID NO: 2, SEQ ID NO: 6, SEQ ID NO: 12,or SEQ ID NO: 14; at least 65%, at least 70%, at least 75%, at least80%, at least 81%, at least 83%, at least 83%, at least 84%, at least85%, at least 86%, at least 87%, at least 88%, at least 89%, at least90%, at least 91%, at least 92%, at least 93%, at least 94%, at least95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%sequence identity to the mature polypeptide of SEQ ID NO: 8 or SEQ IDNO: 10, or at least 75%, at least 80%, at least 81%, at least 83%, atleast 83%, at least 84%, at least 85%, at least 86%, at least 87%, atleast 88%, at least 89%, at least 90%, at least 91%, at least 92%, atleast 93%, at least 94%, at least 95%, at least 96%, at least 97%, atleast 98%, at least 99%, or 100% sequence identity to the maturepolypeptide of SEQ ID NO: 4.

[3] The polypeptide of paragraph 1 or 2, which is encoded by apolynucleotide that hybridizes under medium-high, high, or very highstringency conditions with (i) the mature polypeptide coding sequence ofSEQ ID NO: 1 or the cDNA sequence thereof, SEQ ID NO: 3, SEQ ID NO: 5,SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, or SEQ ID NO: 13 or the cDNAsequence thereof, or (ii) the full-length complement of (i).

[4] The polypeptide of any of paragraphs 1-3, which is encoded by apolynucleotide having at least 60%, at least 65%, at least 70%, at least75%, at least 80%, at least 81%, at least 83%, at least 83%, at least84%, at least 85%, at least 86%, at least 87%, at least 88%, at least89%, at least 90%, at least 91%, at least 92%, at least 93%, at least94%, at least 95%, at least 96%, at least 97%, at least 98%, at least99%, or 100% sequence identity to the mature polypeptide coding sequenceof SEQ ID NO: 1 or the cDNA sequence thereof, SEQ ID NO: 5, SEQ ID NO:11, or SEQ ID NO: 13 or the cDNA sequence thereof; at least 65%, atleast 70%, at least 75%, at least 80%, at least 81%, at least 83%, atleast 83%, at least 84%, at least 85%, at least 86%, at least 87%, atleast 88%, at least 89%, at least 90%, at least 91%, at least 92%, atleast 93%, at least 94%, at least 95%, at least 96%, at least 97%, atleast 98%, at least 99%, or 100% sequence identity to the maturepolypeptide of SEQ ID NO: 7 or SEQ ID NO: 9; or at least 75%, at least80%, at least 81%, at least 83%, at least 83%, at least 84%, at least85%, at least 86%, at least 87%, at least 88%, at least 89%, at least90%, at least 91%, at least 92%, at least 93%, at least 94%, at least95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%sequence identity to the mature polypeptide of SEQ ID NO: 3.

[5] The polypeptide of any of paragraphs 1-4, comprising or consistingof SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO:10, SEQ ID NO: 12, or SEQ ID NO: 14; or the mature polypeptide thereof.

[6] The polypeptide of paragraph 5, wherein the mature polypeptide isamino acids 21 to 344 of SEQ ID NO: 2, amino acids 26 to 400 of SEQ IDNO: 4, amino acids 21 to 389 of SEQ ID NO: 6, amino acids 22 to 405 ofSEQ ID NO: 8, amino acids 20 to 427 of SEQ ID NO: 10, amino acids 18 to267 of SEQ ID NO: 12, or amino acids 21 to 273 of SEQ ID NO: 14.

[7] The polypeptide of any of paragraphs 1-4, which is a variant of themature polypeptide of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ IDNO: 8, SEQ ID NO: 10, SEQ ID NO: 12, or SEQ ID NO: 14 comprising asubstitution, deletion, and/or insertion at one or more positions.

[8] The polypeptide of paragraph 1, which is a fragment of SEQ ID NO: 2,SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12,or SEQ ID NO: 14, wherein the fragment has cellulolytic enhancingactivity.

[9] A composition comprising the polypeptide of any of paragraphs 1-8.

[10] An isolated polynucleotide encoding the polypeptide of any ofparagraphs 1-8.

[11] A nucleic acid construct or expression vector comprising thepolynucleotide of paragraph 10 operably linked to one or more controlsequences that direct the production of the polypeptide in an expressionhost.

[12] A recombinant host cell comprising the polynucleotide of paragraph10 operably linked to one or more control sequences that direct theproduction of the polypeptide.

[13] A method of producing the polypeptide of any of paragraphs 1-8,comprising: (a) cultivating a cell, which in its wild-type form producesthe polypeptide, under conditions conducive for production of thepolypeptide; and (b) recovering the polypeptide.

[14] A method of producing a polypeptide having cellulolytic enhancingactivity, comprising: (a) cultivating the host cell of paragraph 12under conditions conducive for production of the polypeptide; and (b)recovering the polypeptide.

[15] A transgenic plant, plant part or plant cell transformed with apolynucleotide encoding the polypeptide of any of paragraphs 1-8.

[16] A method of producing a polypeptide having cellulolytic enhancingactivity, comprising: (a) cultivating the transgenic plant or plant cellof paragraph 15 under conditions conducive for production of thepolypeptide; and (b) recovering the polypeptide.

[17] A method of producing a mutant of a parent cell, comprisinginactivating a polynucleotide encoding the polypeptide of any ofparagraphs 1-8, which results in the mutant producing less of thepolypeptide than the parent cell.

[18] A mutant cell produced by the method of paragraph 17.

[19] The mutant cell of paragraph 18, further comprising a gene encodinga native or heterologous protein.

[20] A method of producing a protein, comprising: (a) cultivating themutant cell of paragraph 18 or 19 under conditions conducive forproduction of the protein; and (b) recovering the protein.

[21] A double-stranded inhibitory RNA (dsRNA) molecule comprising asubsequence of the polynucleotide of paragraph 10, wherein optionallythe dsRNA is an siRNA or an miRNA molecule.

[22] The double-stranded inhibitory RNA (dsRNA) molecule of paragraph21, which is about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or moreduplex nucleotides in length.

[23] A method of inhibiting the expression of a polypeptide havingcellulolytic enhancing activity in a cell, comprising administering tothe cell or expressing in the cell the double-stranded inhibitory RNA(dsRNA) molecule of paragraph 21 or 22.

[24] A cell produced by the method of paragraph 23.

[25] The cell of paragraph 24, further comprising a gene encoding anative or heterologous protein.

[26] A method of producing a protein, comprising: (a) cultivating thecell of paragraph 24 or 25 under conditions conducive for production ofthe protein; and (b) recovering the protein.

[27] An isolated polynucleotide encoding a signal peptide comprising orconsisting of amino acids 1 to 20 of SEQ ID NO: 2, amino acids 1 to 25of SEQ ID NO: 4, amino acids 1 to 20 of SEQ ID NO: 6, amino acids 1 to21 of SEQ ID NO: 8, amino acids 1 to 19 of SEQ ID NO: 10, amino acids 1to 17 of SEQ ID NO: 12, or amino acids 1 to 20 of SEQ ID NO: 14.

[28] A nucleic acid construct or expression vector comprising a geneencoding a protein operably linked to the polynucleotide of paragraph27, wherein the gene is foreign to the polynucleotide encoding thesignal peptide.

[29] A recombinant host cell comprising a gene encoding a proteinoperably linked to the polynucleotide of paragraph 27, wherein the geneis foreign to the polynucleotide encoding the signal peptide.

[30] A process of producing a protein, comprising: (a) cultivating arecombinant host cell comprising a gene encoding a protein operablylinked to the polynucleotide of paragraph 27, wherein the gene isforeign to the polynucleotide encoding the signal peptide, underconditions conducive for production of the protein; and (b) recoveringthe protein.

[31] A whole broth formulation or cell culture composition comprisingthe polypeptide of any of paragraphs 1-8.

[32] A process for degrading or converting a cellulosic material,comprising: treating the cellulosic material with an enzyme compositionin the presence of the polypeptide having cellulolytic enhancingactivity of any of paragraphs 1-8.

[33] The process of paragraph 32, wherein the cellulosic material ispretreated.

[34] The process of paragraph 32 or 33, wherein the enzyme compositioncomprises one or more enzymes selected from the group consisting of acellulase, a GH61 polypeptide having cellulolytic enhancing activity, ahemicellulase, an esterase, an expansin, a laccase, a ligninolyticenzyme, a pectinase, a peroxidase, a protease, and a swollenin.

[35] The process of paragraph 34, wherein the cellulase is one or moreenzymes selected from the group consisting of an endoglucanase, acellobiohydrolase, and a beta-glucosidase.

[36] The process of paragraph 34, wherein the hemicellulase is one ormore enzymes selected from the group consisting of a xylanase, anacetylxylan esterase, a feruloyl esterase, an arabinofuranosidase, axylosidase, and a glucuronidase.

[37] The process of any of paragraphs 32-36, further comprisingrecovering the degraded cellulosic material.

[38] The process of paragraph 37, wherein the degraded cellulosicmaterial is a sugar.

[39] The process of paragraph 38, wherein the sugar is selected from thegroup consisting of glucose, xylose, mannose, galactose, and arabinose.

[40] A process for producing a fermentation product, comprising: (a)saccharifying a cellulosic material with an enzyme composition in thepresence of the polypeptide having cellulolytic enhancing activity ofany of paragraphs 1-8; (b) fermenting the saccharified cellulosicmaterial with one or more fermenting microorganisms to produce thefermentation product; and (c) recovering the fermentation product fromthe fermentation.

[41] The process of paragraph 40, wherein the cellulosic material ispretreated.

[42] The process of paragraph 40 or 41, wherein the enzyme compositioncomprises one or more enzymes selected from the group consisting of acellulase, a GH61 polypeptide having cellulolytic enhancing activity, ahemicellulase, an esterase, an expansin, a laccase, a ligninolyticenzyme, a pectinase, a peroxidase, a protease, and a swollenin.

[43] The process of paragraph 42, wherein the cellulase is one or moreenzymes selected from the group consisting of an endoglucanase, acellobiohydrolase, and a beta-glucosidase.

[44] The process of paragraph 42, wherein the hemicellulase is one ormore enzymes selected from the group consisting of a xylanase, anacetylxylan esterase, a feruloyl esterase, an arabinofuranosidase, axylosidase, and a glucuronidase.

[45] The process of any of paragraphs 40-44, wherein steps (a) and (b)are performed simultaneously in a simultaneous saccharification andfermentation.

[46] The process of any of paragraphs 40-45, wherein the fermentationproduct is an alcohol, an alkane, a cycloalkane, an alkene, an aminoacid, a gas, isoprene, a ketone, an organic acid, or polyketide.

[47] A process of fermenting a cellulosic material, comprising:fermenting the cellulosic material with one or more fermentingmicroorganisms, wherein the cellulosic material is saccharified with anenzyme composition in the presence of a polypeptide having cellulolyticenhancing activity of any of paragraphs 1-8.

[48] The process of paragraph 47, wherein the fermenting of thecellulosic material produces a fermentation product.

[49] The process of paragraph 48, further comprising recovering thefermentation product from the fermentation.

[50] The process of any of paragraphs 47-49, wherein the cellulosicmaterial is pretreated before saccharification.

[51] The process of any of paragraphs 47-50, wherein the enzymecomposition comprises one or more enzymes selected from the groupconsisting of a cellulase, a GH61 polypeptide having cellulolyticenhancing activity, a hemicellulase, an esterase, an expansin, alaccase, a ligninolytic enzyme, a pectinase, a peroxidase, a protease,and a swollenin.

[52] The process of paragraph 51, wherein the cellulase is one or moreenzymes selected from the group consisting of an endoglucanase, acellobiohydrolase, and a beta-glucosidase.

[53] The process of paragraph 51, wherein the hemicellulase is one ormore enzymes selected from the group consisting of a xylanase, anacetylxylan esterase, a feruloyl esterase, an arabinofuranosidase, axylosidase, and a glucuronidase.

[54] The process of any of paragraphs 47-53, wherein the fermentationproduct is an alcohol, an alkane, a cycloalkane, an alkene, an aminoacid, a gas, isoprene, a ketone, an organic acid, or polyketide.

The invention described and claimed herein is not to be limited in scopeby the specific aspects herein disclosed, since these aspects areintended as illustrations of several aspects of the invention. Anyequivalent aspects are intended to be within the scope of thisinvention. Indeed, various modifications of the invention in addition tothose shown and described herein will become apparent to those skilledin the art from the foregoing description. Such modifications are alsointended to fall within the scope of the appended claims. In the case ofconflict, the present disclosure including definitions will control.

What is claimed is:
 1. A nucleic acid construct comprising apolynucleotide encoding a Family 61 glycoside hydrolase (GH61)polypeptide having cellulolytic enhancing activity, wherein thepolynucleotide is operably linked to one or more heterologous controlsequences that direct the production of the GH61 polypeptide and whereinthe GH61 polypeptide is selected from the group consisting of: (a) aGH61 polypeptide having at least 95% sequence identity to amino acids 21to 344 of SEQ ID NO: 2; (b) a GH61 polypeptide encoded by apolynucleotide having at least 95% sequence identity to nucleotides 61to 1032 of SEQ ID NO: 1 or the cDNA of nucleotides 61 to 1032 of SEQ IDNO: 1; (c) a GH61 polypeptide comprising amino acids 21 to 344 of SEQ IDNO: 2; and (d) a GH61 polypeptide encoded by a polynucleotide comprisingnucleotides 61 to 1032 of SEQ ID NO: 1 or the cDNA of nucleotides 61 to1032 of SEQ ID NO:
 1. 2. An isolated recombinant host cell comprisingthe nucleic acid construct of claim
 1. 3. A method of producing a GH61polypeptide having cellulolytic enhancing activity, said methodcomprising: (a) cultivating the recombinant host cell of claim 2 underconditions conducive for production of the GH61 polypeptide; and (b)recovering the GH61 polypeptide.
 4. A transgenic plant, plant part orplant cell transformed with the nucleic acid construct of claim 1comprising a polynucleotide encoding the GH61 polypeptide havingcellulolytic enhancing activity.
 5. A method of producing a GH61polypeptide having cellulolytic enhancing activity, said methodcomprising: (a) cultivating the transgenic plant or plant cell of claim4 under conditions conducive for production of the GH61 polypeptide; and(b) recovering the GH61 polypeptide.
 6. An isolated polynucleotidecomprising a gene encoding a protein and a nucleic acid encoding asignal peptide comprising amino acids 1 to 20 of SEQ ID NO: 2, whereinsaid gene is foreign to and operably linked to the nucleic acid encodingthe signal peptide.
 7. An isolated recombinant host cell comprising thepolynucleotide of claim
 6. 8. A process of producing a protein, saidprocess comprising: (a) cultivating the recombinant host cell of claim 7under conditions conducive for production of the protein; and (b)recovering the protein.
 9. A process for degrading or converting acellulosic material, said process comprising: treating the cellulosicmaterial with an enzyme composition comprising a GH61 polypeptide havingcellulolytic enhancing activity, and recovering the degraded orconverted cellulosic material, wherein the GH61 polypeptide is selectedfrom the group consisting of: (a) a GH61 polypeptide having at least 95%sequence identity to amino acids 21 to 344 of SEQ ID NO: 2; (b) a GH61polypeptide encoded by a polynucleotide having at least 95% sequenceidentity to nucleotides 61 to 1032 of SEQ ID NO: 1 or the cDNA ofnucleotides 61 to 1032 of SEQ ID NO: 1; (c) a GH61 polypeptidecomprising amino acids 21 to 344 of SEQ ID NO: 2; and (d) a GH61polypeptide encoded by a polynucleotide comprising nucleotides 61 to1032 of SEQ ID NO: 1 or the cDNA of nucleotides 61 to 1032 of SEQ IDNO:
 1. 10. A process for producing a fermentation product, said processcomprising: (a) saccharifying a cellulosic material with an enzymecomposition comprising a GH61 polypeptide having cellulolytic enhancingactivity, wherein the GH61 polypeptide is selected from the groupconsisting of: (1) a GH61 polypeptide having at least 95% sequenceidentity to amino acids 21 to 344 of SEQ ID NO: 2; (2) a GH61polypeptide encoded by a polynucleotide having at least 95% sequenceidentity to nucleotides 61 to 1032 of SEQ ID NO: 1 or the cDNA ofnucleotides 61 to 1032 of SEQ ID NO: 1; (3) a GH61 polypeptidecomprising amino acids 21 to 344 of SEQ ID NO: 2; and (4) a GH61polypeptide encoded by a polynucleotide comprising nucleotides 61 to1032 of SEQ ID NO: 1 or the cDNA of nucleotides 61 to 1032 of SEQ ID NO:1; (b) fermenting the saccharified cellulosic material with one or morefermenting microorganisms to produce the fermentation product; and (c)recovering the fermentation product from the fermentation.
 11. A processof fermenting a cellulosic material, said process comprising: fermentingthe cellulosic material with one or more fermenting microorganisms,wherein the cellulosic material is saccharified with an enzymecomposition comprising a GH61 polypeptide having cellulolytic enhancingactivity, wherein the fermenting of the cellulosic material produces afermentation product, and recovering the fermentation product from thefermentation, and wherein the GH61 polypeptide is selected from thegroup consisting of: (a) a GH61 polypeptide having at least 95% sequenceidentity to amino acids 21 to 344 of SEQ ID NO: 2; (b) a GH61polypeptide encoded by a polynucleotide having at least 95% sequenceidentity to nucleotides 61 to 1032 of SEQ ID NO: 1 or the cDNA ofnucleotides 61 to 1032 of SEQ ID NO: 1; (c) a GH61 polypeptidecomprising amino acids 21 to 344 of SEQ ID NO: 2; and (d) a GH61polypeptide encoded by a polynucleotide comprising nucleotides 61 to1032 of SEQ ID NO: 1 or the cDNA of nucleotides 61 to 1032 of SEQ IDNO:
 1. 12. The nucleic acid construct of claim 1, wherein the GH61polypeptide has at least 96% sequence identity to amino acids 21 to 344of SEQ ID NO:
 2. 13. The nucleic acid construct of claim 1, wherein theGH61 polypeptide has at least 97% sequence identity to amino acids 21 to344 of SEQ ID NO:
 2. 14. The nucleic acid construct of claim 1, whereinthe GH61 polypeptide has at least 98% sequence identity to amino acids21 to 344 of SEQ ID NO:
 2. 15. The nucleic acid construct of claim 1,wherein the GH61 polypeptide has at least 99% sequence identity to aminoacids 21 to 344 of SEQ ID NO:
 2. 16. The nucleic acid construct of claim1, wherein the GH61 polypeptide comprises amino acids 21 to 344 of SEQID NO:
 2. 17. The nucleic acid construct of claim 1, wherein the GH61polypeptide is encoded by a polynucleotide having at least 96% sequenceidentity to nucleotides 61 to 1032 of SEQ ID NO: 1 or the cDNA ofnucleotides 61 to 1032 of SEQ ID NO:
 1. 18. The nucleic acid constructof claim 1, wherein the GH61 polypeptide is encoded by a polynucleotidehaving at least 97% sequence identity to nucleotides 61 to 1032 of SEQID NO: 1 or the cDNA of nucleotides 61 to 1032 of SEQ ID NO:
 1. 19. Thenucleic acid construct of claim 1, wherein the GH61 polypeptide isencoded by a polynucleotide having at least 98% sequence identity tonucleotides 61 to 1032 of SEQ ID NO: 1 or the cDNA of nucleotides 61 to1032 of SEQ ID NO:
 1. 20. The nucleic acid construct of claim 1, whereinthe GH61 polypeptide is encoded by a polynucleotide having at least 99%sequence identity to nucleotides 61 to 1032 of SEQ ID NO: 1 or the cDNAof nucleotides 61 to 1032 of SEQ ID NO:
 1. 21. The nucleic acidconstruct of claim 1, wherein the GH61 polypeptide is encoded by apolynucleotide comprising nucleotides 61 to 1032 of SEQ ID NO: 1 or thecDNA of nucleotides 61 to 1032 of SEQ ID NO:
 1. 22. The process of claim9, wherein the GH61 polypeptide has at least 96% sequence identity toamino acids 21 to 344 of SEQ ID NO:
 2. 23. The process of claim 9,wherein the GH61 polypeptide has at least 97% sequence identity to aminoacids 21 to 344 of SEQ ID NO:
 2. 24. The process of claim 9, wherein theGH61 polypeptide has at least 98% sequence identity to amino acids 21 to344 of SEQ ID NO:
 2. 25. The process of claim 9, wherein the GH61polypeptide has at least 99% sequence identity to amino acids 21 to 344of SEQ ID NO:
 2. 26. The process of claim 9, wherein the GH61polypeptide comprises amino acids 21 to 344 of SEQ ID NO:
 2. 27. Theprocess of claim 9, wherein the GH61 polypeptide is encoded by apolynucleotide having at least 96% sequence identity to nucleotides 61to 1032 of SEQ ID NO: 1 or the cDNA of nucleotides 61 to 1032 of SEQ IDNO:
 1. 28. The process of claim 9, wherein the GH61 polypeptide isencoded by a polynucleotide having at least 97% sequence identity tonucleotides 61 to 1032 of SEQ ID NO: 1 or the cDNA of nucleotides 61 to1032 of SEQ ID NO:
 1. 29. The process of claim 9, wherein the GH61polypeptide is encoded by a polynucleotide having at least 98% sequenceidentity to nucleotides 61 to 1032 of SEQ ID NO: 1 or the cDNA ofnucleotides 61 to 1032 of SEQ ID NO:
 1. 30. The process of claim 9,wherein the GH61 polypeptide is encoded by a polynucleotide having atleast 99% sequence identity to nucleotides 61 to 1032 of SEQ ID NO: 1 orthe cDNA of nucleotides 61 to 1032 of SEQ ID NO:
 1. 31. The process ofclaim 9, wherein the GH61 polypeptide is encoded by a polynucleotidecomprising nucleotides 61 to 1032 of SEQ ID NO: 1 or the cDNA ofnucleotides 61 to 1032 of SEQ ID NO:
 1. 32. The process of claim 9,wherein the cellulosic material is pretreated.
 33. The process of claim9, wherein the enzyme composition further comprises one or more enzymesselected from the group consisting of a cellulase, a GH61 polypeptidehaving cellulolytic enhancing activity, a hemicellulase, an esterase, anexpansin, a laccase, a ligninolytic enzyme, a pectinase, a peroxidase, aprotease, and a swollenin.
 34. The process of claim 9, furthercomprising recovering the degraded or converted cellulosic material. 35.The process of claim 34, wherein the degraded or converted cellulosicmaterial is a sugar.
 36. The process of claim 35, wherein the sugar isselected from the group consisting of glucose, xylose, mannose,galactose, and arabinose.
 37. The process of claim 10, wherein the GH61polypeptide has at least 97% sequence identity to amino acids 21 to 344of SEQ ID NO:
 2. 38. The process of claim 10, wherein the GH61polypeptide has at least 98% sequence identity to amino acids 21 to 344of SEQ ID NO:
 2. 39. The process of claim 10, wherein the GH61polypeptide has at least 99% sequence identity to amino acids 21 to 344of SEQ ID NO:
 2. 40. The process of claim 10, wherein the GH61polypeptide comprises amino acids 21 to 344 of SEQ ID NO:
 2. 41. Theprocess of claim 10, wherein the GH61 polypeptide is encoded by apolynucleotide having at least 96% sequence identity to nucleotides 61to 1032 of SEQ ID NO: 1 or the cDNA of nucleotides 61 to 1032 of SEQ IDNO:
 1. 42. The process of claim 10, wherein the GH61 polypeptide isencoded by a polynucleotide having at least 97% sequence identity tonucleotides 61 to 1032 of SEQ ID NO: 1 or the cDNA of nucleotides 61 to1032 of SEQ ID NO:
 1. 43. The process of claim 10, wherein the GH61polypeptide is encoded by a polynucleotide having at least 98% sequenceidentity to nucleotides 61 to 1032 of SEQ ID NO: 1 or the cDNA ofnucleotides 61 to 1032 of SEQ ID NO:
 1. 44. The process of claim 10,wherein the GH61 polypeptide is encoded by a polynucleotide having atleast 99% sequence identity to nucleotides 61 to 1032 of SEQ ID NO: 1 orthe cDNA of nucleotides 61 to 1032 of SEQ ID NO:
 1. 45. The process ofclaim 10, wherein the GH61 polypeptide is encoded by a polynucleotidecomprising nucleotides 61 to 1032 of SEQ ID NO: 1 or the cDNA ofnucleotides 61 to 1032 of SEQ ID NO:
 1. 46. The process of claim 10,wherein the cellulosic material is pretreated.
 47. The process of claim10, wherein the enzyme composition further comprises one or more enzymesselected from the group consisting of a cellulase, a GH61 polypeptidehaving cellulolytic enhancing activity, a hemicellulase, an esterase, anexpansin, a laccase, a ligninolytic enzyme, a pectinase, a peroxidase, aprotease, and a swollenin.
 48. The process of claim 10, wherein steps(a) and (b) are performed simultaneously in a simultaneoussaccharification and fermentation.
 49. The process of claim 10, whereinthe fermentation product is an alcohol, an alkane, a cycloalkane, analkene, an amino acid, a gas, isoprene, a ketone, an organic acid, orpolyketide.
 50. The process of claim 11, wherein the GH61 polypeptidehas at least 97% sequence identity to amino acids 21 to 344 of SEQ IDNO:
 2. 51. The process of claim 11, wherein the GH61 polypeptide has atleast 98% sequence identity to amino acids 21 to 344 of SEQ ID NO: 2.52. The process of claim 11, wherein the GH61 polypeptide has at least99% sequence identity to amino acids 21 to 344 of SEQ ID NO:
 2. 53. Theprocess of claim 11, wherein the GH61 polypeptide comprises amino acids21 to 344 of SEQ ID NO:
 2. 54. The process of claim 11, wherein the GH61polypeptide is encoded by a polynucleotide having at least 96% sequenceidentity to nucleotides 61 to 1032 of SEQ ID NO: 1 or the cDNA ofnucleotides 61 to 1032 of SEQ ID NO:
 1. 55. The process of claim 11,wherein the GH61 polypeptide is encoded by a polynucleotide having atleast 97% sequence identity to nucleotides 61 to 1032 of SEQ ID NO: 1 orthe cDNA of nucleotides 61 to 1032 of SEQ ID NO:
 1. 56. The process ofclaim 11, wherein the GH61 polypeptide is encoded by a polynucleotidehaving at least 98% sequence identity to nucleotides 61 to 1032 of SEQID NO: 1 or the cDNA of nucleotides 61 to 1032 of SEQ ID NO:
 1. 57. Theprocess of claim 11, wherein the GH61 polypeptide is encoded by apolynucleotide having at least 99% sequence identity to nucleotides 61to 1032 of SEQ ID NO: 1 or the cDNA of nucleotides 61 to 1032 of SEQ IDNO:
 1. 58. The process of claim 11, wherein the GH61 polypeptide isencoded by a polynucleotide comprising nucleotides 61 to 1032 of SEQ IDNO: 1 or the cDNA of nucleotides 61 to 1032 of SEQ ID NO:
 1. 59. Theprocess of claim 11, wherein the cellulosic material is pretreatedbefore saccharification.
 60. The process of claim 11, wherein the enzymecomposition further comprises one or more enzymes selected from thegroup consisting of a cellulase, a GH61 polypeptide having cellulolyticenhancing activity, a hemicellulase, an esterase, an expansin, alaccase, a ligninolytic enzyme, a pectinase, a peroxidase, a protease,and a swollenin.
 61. The process of claim 11, wherein the fermentationproduct is an alcohol, an alkane, a cycloalkane, an alkene, an aminoacid, a gas, isoprene, a ketone, an organic acid, or polyketide.
 62. Theprocess of claim 10, wherein the GH61 polypeptide has at least 96%sequence identity to amino acids 21 to 344 of SEQ ID NO:2.
 63. Theprocess of claim 11, wherein the GH61 polypeptide has at least 96%sequence identity to amino acids 21 to 344 of SEQ ID NO:2.